Method for producing transgenic birds and fish

ABSTRACT

The present invention relates to methods for producing transgenic animals, particularly transgenic birds and fish, using retroviral constructs engineered to carry the transgene(s) of interest.

REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. §119(e)to U.S. Provisional Application No. 60/322,031, filed Sep. 13, 2001 andto U.S. Provisional Application No. 60/347,782, filed Jan. 9, 2002.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under GrantNumber GM39458 awarded by the National Institutes of Health. The UnitedStates Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to methods for generatingtransgenic animals, particularly transgenic birds and fish, using viralconstructs engineered to carry the transgene(s) of interest.

[0005] 2. Description of the Related Art

[0006] Early transgenic experiments used an oncoretrovirus to introducethe gene of interest into embryonic cells (Jaenisch Proc. Natl. Acad.Sci. USA 73:1260-1264 (1976)). In a typical experiment an engineeredMoloney strain of mouse leukemia virus (MoMLV) was injected into theblastocyst cavity of mice. While the transgene was often integrated intothe genome of the resulting mice, no gene expression could be detected.

[0007] Today, the majority of transgenic animals are made using directinjection technology (Gordon and Ruddle Science 214:1244-1246 (1981)).Briefly, a DNA construct carrying the gene of interest is injecteddirectly into the pronucleus of a single-cell zygote. The cell is thenimplanted into a pseudo-pregnant female and the resulting progeny isanalyzed for expression of the gene.

[0008] While this method achieves both integration and expression of thetransgene, there are a number of significant drawbacks to the directinjection technique. First, in order to carry out the technique it isnecessary to inject DNA directly into the pronucleus. This is possiblein some specific strains of mice, such as Black6 x BDA, because the malepronucleus is visible. However, in other animals and other strains ofmice the pronucleus is less visible, making the technique extremelydifficult. Further, the injection requires the assistance of a skilledtechnician and a significant investment in equipment; micromanipulatorsare necessary to hold the cell and the injection pipette and a pressuresource is required that can deliver picoliter amounts of DNA solution.

[0009] A second equally significant problem with the direct injectionmethod is the low percentage of injected zygotes that produce transgenicanimals. The injection pipette must go through the zona pellucida, thecell membrane and the nuclear envelope. Thus only 80-90% of mouse cellssurvive the injection. Other animal cells are less hardy and thesurvival rates are somewhat lower, with about 60% survival for rats and40-50% for cows. In mice, of the original zygotes, about 25% aresuccessfully injected and implanted in a pseudopregnant female. About20% of the resulting animals have the transgene integrated into theirgenome. Of these, about 20% will express the gene. However, even if theanimals express the gene, it is possible that the expression patternwill not be useful. Thus, only about 1% of injected zygotes result intransgenic animals that express the gene of interest. This lowefficiency of transgenesis is particularly troubling for larger animals,such as pigs, cows or goats, in which obtaining large numbers of embryosis not possible (see, e.g., Wall et al. J. Cell. Biochem. 49:113(1992)).

[0010] In addition, direct pronuclear injection is not possible for manytypes of animals, including birds.

SUMMARY OF THE INVENTION

[0011] One aspect of the present invention concerns a method ofproducing a transgenic animal, preferably a transgenic bird or atransgenic fish. In one embodiment a method is provided for producing atransgenic bird, the method comprising transfecting a packaging cellline with a retroviral construct, recovering recombinant retrovirus, andinfecting a fertilized bird egg with the recombinant retrovirus.

[0012] In one embodiment infecting a bird egg comprises contacting theembryonic blastodisc of the bird egg with the retroviral particles.

[0013] The retroviral construct preferably comprises the R and U5sequences from a 5′ lentiviral long terminal repeat (LTR) and aself-inactivating lentiviral 3′ LTR. Further, the self-inactivating 3′LTR preferably comprises a U3 element with a deletion of its enhancersequence. In one embodiment the LTR sequences are from HIV.

[0014] In one embodiment the retroviral construct comprises a gene thatis desirably expressed in the transgenic bird. In this embodiment theretroviral construct may also comprise an internal promoter and/orenhancer. In one embodiment the internal promoter is ubiquitous. Theubiquitous promoter may be any ubiquitous promoter known in the art. Forexample, the ubiquitous promoter may be selected from the groupconsisting of the ubiquitin promoter, the CMV β-actin promoter and thepgk promoter. In another embodiment the internal promoter istissue-specific. The tissue specific promoter may be any tissue specificpromoter known in the art, for example, a promoter selected from thegroup consisting of the lck promoter, the myogenin promoter and the thy1promoter.

[0015] In addition, the recombinant retrovirus may be pseudotyped. Thus,in one embodiment the recombinant retrovirus is pseudotyped with thevesicular stomatitis virus envelope glycoprotein.

[0016] The viral construct may comprise one or more additional geneticelements. In one embodiment the viral construct comprises a promoteroperably linked to the R and U5 5′ LTR sequences, preferably a CMVpromoter sequence. An enhancer, preferably a CMV enhancer sequence, mayalso be included in the viral construct.

[0017] In another embodiment the viral construct comprises a woodchuckhepatitis virus enhancer element sequence. In yet another embodiment theviral construct comprises a tRNA amber suppressor sequence.

[0018] The viral construct may additionally comprise a reporter geneoperably linked to the internal promoter. The reporter gene may encodebe a fluorescent protein, preferably green fluorescent protein.

[0019] In another aspect the invention concerns a method of producing atransgenic bird comprising opening a window in the shell of a fertilizedbird egg, injecting modified retrovirus into the space between theperivitelline membrane and the embryonic blastodisc and incubating theembryo until hatching. The modified retrovirus is preferably a modifiedlentivirus. The modified lentivirus is preferably produced bytransfecting a packaging cell line with a viral construct. In oneembodiment the viral construct comprises the R and U5 sequences from alentiviral 5′ LTR, an internal promoter, a gene of interest and a selfinactivating lentiviral 3′ LTR.

[0020] In yet another aspect the invention concerns a transgenic birdmade by any of the disclosed methods. Thus, the transgenic birdpreferably comprises one or more cells, preferably germ cells, thatcomprise proviral DNA. The proviral DNA may comprise a self-inactivatinglentiviral 3′ LTR, such as a self-inactivating HIV 3′ LTR. Inparticular, the self-inactivating 3′ LTR may have a deletion of itsenhancer sequence.

[0021] In another embodiment a transgenic fish is produced by a methodthat comprises transfecting a packaging cell line with a viralconstruct, recovering recombinant retroviral particles, and infecting afish egg with the recombinant retroviral particles. Preferably the viralconstruct comprises the R and U5 sequences from a 5′ lentiviral LTR anda self-inactivating 3′ lentiviral LTR. Infecting the fish egg preferablycomprises delivering the retroviral particles to the space between thechorion and the cell membrane of the fish egg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1A is a diagram of the FUGW viral construct. FIG. 1B is adiagram of the provirus that is integrated into the host genome afterinfection with recombinant virus prepared with the FUGW viral constructof FIG. 1A.

[0023]FIG. 2 is a Southern blot analysis of proviral transgeneinsertions in the founder generation of mice generated by injectingrecombinant lentivirus into the perivitelline space of one-cell embryos.Genomic DNA from each animal was digested with either PstI (left) orBamHI (right), and probed with a GFP+WRE sequence. All PstI and BamHIsites in the provirus are located 5′ to the GFP gene. Plus signs aboveeach lane indicate GFP expression in the animal detectable by viewingunder conventional epifluorescence.

[0024]FIG. 3 is a Southern blot analysis of proviral transgeneinsertions in the founder generation of a second group of mice generatedby injecting recombinant lentivirus into the perivitelline space ofone-cell embryos. Genomic DNA from each animal was digested with BamHIand probed with a GFP+WRE sequence. All BamHI sites in the provirus arelocated 5′ to the GFP gene. Plus signs above each lane indicate GFPexpression in the animal detectable by viewing under conventionalfluorescence. Of the 56 founder animals in this experiment, 45 or 80.4%are transgenic. Of these 45 transgenic animals, 41 or 91.1% express GFPat detectable levels. Lanes marked “C” are positive plasmid controls.

[0025]FIG. 4 shows ubiquitous GFP expression in rats derived from thedelivery of modified lentivirus to single-cell embryos in vitro. FIG. 4Ashows brightfield (BF) and fluorescent images of the paws of newbornrats derived from a FUGW-injected embryo. Pup R4, carrying 4 copies ofthe proviral insert, expresses GFP in the paw, as well as all othertissues and organs examined. A littermate (R3) carrying no transgene isincluded for comparison. FIG. 4B shows a Southern blot analysis ofproviral insertions in rats generated by injection of FUGW lentivirusinto the perivitelline space of single-cell embryos. Genomic DNA wasdigested with PstI and hybridized with a GFP+WRE probe. Plus signs aboveeach lane indicate GFP expression in the animal detectable by directviewing under a fluorescent microscope.

[0026]FIG. 5 is a Southern blot analysis of proviral transgeneinsertions in the founder generation of mice generated by incubatingdenuded embryos in media comprising recombinant lentivirus produced withthe FUGW viral construct. Genomic DNA from each animal was digested withPstI and probed with a GFP+WRE sequence. All PstI sites in the provirusare located 5′ to the GFP gene. The ratios above the lanes indicate thedilution of the virus from 1×10⁶ pfu/μl.

[0027]FIG. 6 shows GFP expression in major tissues and organs of afounder mouse. The mouse was perfused intracardially with PBS, pH 7.4,and then 3% paraformaldehyde, and viewed immediately under a fluorescentdissecting microscope. The particular mouse shown was generated byco-incubation of the denuded embryo with the lentiviral suspension andcontains 8 proviral insertions. A wildtype animal, identically perfusedand photographed is included for comparison.

[0028]FIG. 7 is a Southern blot analysis of proviral transgeneinsertions in the F1 progeny of founder transgenic mice, showing thatthe F1 progeny inherit the proviral transgene in a Mendelian fashion.The founder mice were generated by injection of FUGW lentivirus into theperivitelline space of single-cell embryos. Genomic DNA from each animalwas digested with BamHI and probed wiht GFP+WRE sequence. All BamHIsites are located 5′ to the GFP gene. The first numbered lane in eachgroup is the P0 founder animal, while the lettered lanes representprogeny resulting from outcrossing that founder animal to a wildtypeanimal. Plus signs above each lane indicate GFP expression in thatanimal detectable by direct viewing of the live animal under aconventional epifluoresence microscope.

[0029]FIG. 8 shows that transgenic mice give rise to transgenic progenythat express the transgene. This indicates that the transgene can gothrough an entire round of gametogenesis and development without beingsilenced. Expression of the transgene was determined based on GFPexpression in the newborn pup. The pup imaged here is descended from ananimal with 10 proviral insertions.

[0030]FIG. 9 is a Southern blot analysis of proviral transgeneinsertions in the founder generation of mice generated using alentiviral vector containing the myogenin promoter driving ahistone2B-GFP fusion. Embryos were recovered from the uterus atembryonic day 11.5 (“E11.5”). The litter consisted of 6 animals, all ofwhich were transgenic. Genomic DNA from each animal was digested withBamHI and probed with a GFP+WRE sequence. A BamHI site is located withinthe histone2B gene, 5′ of the GFP sequence in the provirus. Plus signsabove the lanes indicate positive plasmid controls. Animals 5 and 6 werepositive for tissue-specific GFP expression at embryonic day 11.5 whenviewed as a whole mount under an inverted fluorescent microscope, andanimal 5 expressed more highly than animal 6.

[0031]FIG. 10 shows the GFP expression pattern in an E11.5 mouse embryoderived from the perivitelline space injection of lentivirus carrying ahistone2B-GFP fusion construct under the control of the myogeninpromoter (Yee et al. Genes and Dev. 6:1277-1289 (1993)). GFP expressionis localized to the somites and can be seen in the emerging muscles inthe limb buds, eye and jaw.

[0032]FIG. 11 shows immunofluorescence with an antibody against GFP in across-section through an E11.5 embryo carrying the myogenin promoterdriving GFP expression. Embryos were derived from single-cell zygotesinjected with recombinant lentivirus in the perivitelline space. Embryoswere fixed in 3% paraformaldehyde, cryoprotected in 30% sucroseovernight and 30 μm sections were cut on a cryostat. Sections wereincubated with a polyclonal antibody against GFP and probed witha-rabbit secondary antibody conjugated to a rhodamine fluorophore.Images on the left are sections as viewed under a rhodamine filter,while images on the right show the nuclear counterstain Hoechst-33342for each corresponding section. The animal carried 6 proviral insertionsof the myogenin-GFP construct. Specific staining of somite tissues canbe seen, with the exclusion of the stain from flanking skin and bonetissues.

[0033]FIG. 12 also shows immunofluorescence with an antibody against GFPin cross sections of an E11.5 embryo carrying a myogenin promoterdriving H2B-GFP. Lack of staining in the viscera is noteworthy.

[0034]FIG. 13 also shows immunofluorescence with an antibody against GFPin cross sections of an E11.5 embryo carrying a myogenin promoterdriving H2B-GFP. Specific staining of somites on either side of theneural tube can be visualized.

[0035]FIG. 14 shows H2B-GFP expression in the extraembryonic tissue ofdeveloping zebra finch.

[0036]FIG. 15 shows H2B-GFP expression inside of the zebra finch embryo.

[0037]FIG. 16 shows the nucleotide sequence of GFP.

[0038]FIG. 17 shows the nucleotide sequence of H2B-GFP.

[0039]FIG. 18A shows the nucleotide sequence of HIV NL4.3 flap and 18Bshows the nucleotide sequence of WRE.

[0040]FIG. 19A shows the nucleotide sequence of the myogenin promoterand 19B shows a partial nucleotide sequence of the Lck promoter.

[0041]FIG. 20 shows the nucleotide sequence of the human ubiquitinpromoter.

[0042]FIG. 21 shows the nucleotide sequence of the HIV-1flap+ubiquitin+GFP+WRE construct.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0043] Retroviruses are enveloped RNA viruses that are capable ofinfecting animal cells. When a retrovirus infects a cell, its RNA genomeis converted into a double-stranded linear DNA form by reversetranscription. The DNA form is integrated into the host cell genome as aprovirus. The present invention is based on the discovery thatrecombinant retroviruses can be used to create transgenic animals.Transgenic animals resulting from the methods of the present inventionhave one or more copies of the transgene of interest integrated in theirgenome.

[0044] Previous transgenic technology is not commercially practical inlarger animals, such as monkeys, dogs, poultry, cows, pigs or sheep.Furthermore, previous transgenic methods are not applicable to poultry.Thus, the methods of the present invention will find great commercialapplication, for example in biotechnology and agriculture. The presentmethods may be used to introduce the gene of choice into animals inorder to confer upon them desired attributes. For example, the describedmethods may be used to confer disease resistance. In biotechnology, theability to rapidly develop large numbers of transgenic animals,particular higher order animals such as monkeys, will allow for theanalysis of gene function and the evaluation of compounds thatpotentially modulate gene expression, protein function, or are useful intreating a disease or disorder. Two types of assays in which the methodsof the present invention are particularly useful are gene trap assaysand large-scale mutagenesis screens, each of which is described below.

[0045] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Any methods, devicesand materials similar or equivalent to those described herein can beused in the practice of this invention.

[0046] By “transgene” is meant any nucleotide or DNA sequence that isintegrated into one or more chromosomes of a host cell by humanintervention, such as by the methods of the present invention. In oneembodiment the transgene comprises a “gene of interest.” A “gene ofinterest” is a nucleic acid sequence that encodes a protein or othermolecule that is desirable for integration and/or expression in a hostcell. In this embodiment the gene of interest is generally operativelylinked to other sequences that are useful for obtaining the desiredexpression of the gene of interest, such as transcriptional regulatorysequences. In another embodiment the transgene can be a DNA sequencethat is used to mark the chromosome where it has integrated. In thissituation, the transgene does not have to comprise a gene that encodes aprotein that can be expressed. This use of the transgene as a moleculartag has numerous applications, for example for mutagenesis studies asdescribed below.

[0047] The term “transgenic” is used herein to describe the property ofharboring a transgene. For instance, a “transgenic organism” is anyanimal, including mammals, fish, birds and amphibians, in which one ormore of the cells of the animal contain nucleic acid introduced by wayof human intervention, such as by the methods described herein. In thetypical transgenic animal, the transgene causes the cell to express oroverexpress a recombinant protein. However for some applications, suchas the mutagenesis studies described below, it is not necessary ordesirable for the transgenic organism to express a recombinant protein.

[0048] The terms “founder,” “founder animal” and “founder line” refer tothose animals that are mature products of the embryos or oocytes towhich the transgene was added, i.e. those animals that grew from theembryos or oocytes into which DNA was inserted.

[0049] The terms “progeny” and “progeny of the transgenic animal” referto any and all offspring of every generation subsequent to theoriginally transformed animal.

[0050] The term “animal” is used in its broadest sense and refers to allanimals including mammals, birds, fish, reptiles and amphibians.

[0051] The term “mammal” refers to all members of the class Mammalia andincludes any animal classified as a mammal, including humans, domesticand farm animals, and zoo, sports or pet animals, such as mouse, rabbit,pig, sheep, goat, cattle and higher primates.

[0052] The term “oocyte” refers to a female gamete cell and includesprimary oocytes, secondary oocytes and mature, unfertilized ovum. Asused herein, the term “egg” when used in reference to a mammalian egg,means an oocyte surrounded by a zona pellucida. The term “zygote” refersto a fertilized ovum. The term “embryo” broadly refers to an animal inthe early stages of development.

[0053] “Perivitelline space” refers to the space located between thezona pellucida and the cell membrane of a mammalian egg or embryoniccell.

[0054] “Target cell” or “host cell” means a cell that is to betransformed using the methods and compositions of the invention.

[0055] “Lentivirus” refers to a genus of retroviruses that are capableof infecting dividing and non-dividing cells. Several examples oflentiviruses include HIV (human immunodeficiency virus; including HIVtype 1, and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates.

[0056] A lentiviral genome is generally organized into a 5′ longterminal repeat (LTR), the gag gene, the pol gene, the env gene, theaccessory genes (nef, vif, vpr, vpu) and a 3′ LTR. The viral LTR isdivided into three regions called U3, R and U5. The U3 region containsthe enhancer and promoter elements. The U5 region contains thepolyadenylation signals. The R (repeat) region separates the U3 and U5regions and transcribed sequences of the R region appear at both the 5′and 3′ ends of the viral RNA. See, for example, “RNA Viruses: APractical Approach” (Alan J. Cann, Ed., Oxford University Press,(2000)), O Narayan and Clements J. Gen. Virology 70:1617-1639 (1989),Fields et al. Fundamental Virology Raven Press. (1990), Miyoshi H,Blomer U, Takahashi M, Gage F H, Verma I M. J Virol. 72(10):8150-7(1998), U.S. Pat. No. 6,013,516.

[0057] “Virion,” “viral particle” and “retroviral particle” are usedherein to refer to a single virus comprising an RNA genome, pol genederived proteins, gag gene derived proteins and a lipid bilayerdisplaying an envelope (glyco)protein. The RNA genome is usually arecombinant RNA genome and thus may contain an RNA sequence that isexogenous to the native viral genome. The RNA genome may also comprise adefective endogenous viral sequence.

[0058] A “pseudotyped” retrovirus is a retroviral particle having anenvelope protein that is from a virus other than the virus from whichthe RNA genome is derived. The envelope protein may be from a differentretrovirus or from a non-retroviral virus. A preferred envelope proteinis the vesicular stomatitius virus G (VSV G) protein. However, toeliminate the possibility of human infection, viruses can alternativelybe pseudotyped with ecotropic envelope protein that limit infection to aspecific species, such as mice or birds.

[0059] The term “provirus” is used to refer to a duplex DNA sequencepresent in a eukaryotic chromosome that corresponds to the genome of anRNA retrovirus. The provirus may be transmitted from one cell generationto the next without causing lysis or destruction of the host cell.

[0060] A “self-inactivating 3′ LTR” is a 3′ long terminal repeat (LTR)that contains a mutation, substitution or deletion that prevents the LTRsequences from driving expression of a downstream gene. A copy of the U3region from the 3′ LTR acts as a template for the generation of bothLTR's in the integrated provirus. Thus, when the 3′ LTR with aninactivating deletion or mutation integrates as the 5′ LTR of theprovirus, no transcription from the 5′ LTR is possible. This eliminatescompetition between the viral enhancer/promoter and any internalenhancer/promoter. Self-inactivating 3′ LTRs are described, for example,in Zufferey et al. J Virol. 72:9873-9880 (1998), Miyoshi et al. J Virol.72:8150-8157 and Iwakuma et al. Virology 261:120-132 (1999).

[0061] In one aspect of the invention, a recombinant retrovirus is usedto deliver a transgene of interest to a cell, preferably an oocyte or anembryonic cell, more preferably a one-cell embryo. The transgene, andany associated genetic elements, are thus integrated into the genome ofthe host cell as a provirus. The cell may then be allowed to developinto a transgenic animal.

[0062] The recombinant retrovirus used to deliver the transgene ispreferably a modified lentivirus, and thus is able to infect bothdividing and non-dividing cells. The recombinant retrovirus preferablycomprises a modified lentiviral genome that includes the transgene.Further, the modified lentiviral genome preferably lacks endogenousgenes for proteins required for viral replication, thus preventingreplication in the transgenic animal. The required proteins are providedin trans in the packaging cell line during production of the recombinantretrovirus, as described below.

[0063] In the preferred embodiment the transgene is incorporated into aviral construct that comprises an intact retroviral 5′ LTR and aself-inactivating 3′ LTR. The viral construct is preferably introducedinto a packaging cell line that packages viral genomic RNA based on theviral construct into viral particles with the desired host specificity.Viral particles are collected and allowed to infect the host cell. Eachof these aspects is described in detail below.

[0064] The Viral Construct

[0065] The viral construct is a nucleotide sequence that comprisessequences necessary for the production of recombinant retrovirus in apackaging cell. In one embodiment the viral construct additionallycomprises genetic elements that allow for the desired expression of agene of interest in the host.

[0066] Generation of the viral construct can be accomplished using anysuitable genetic engineering techniques well known in the art,including, without limitation, the standard techniques of restrictionendonuclease digestion, ligation, transformation, plasmid purification,and DNA sequencing, for example as described in Sambrook et al.(Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, N.Y. (1989)), Coffin et al. (Retroviruses. Cold Spring HarborLaboratory Press, N.Y. (1997)) and “RNA Viruses: A Practical Approach”(Alan J. Cann, Ed., Oxford University Press, (2000)).

[0067] The viral construct may incorporate sequences from the genome ofany known organism. The sequences may be incorporated in their nativeform or may be modified in any way. For example, the sequences maycomprise insertions, deletions or substitutions. In the preferredembodiment the viral construct comprises sequences from a lentivirusgenome, such as the HIV genome or the SIV genome.

[0068] The viral construct preferably comprises sequences from the 5′and 3′ LTRs of a lentivirus. More preferably the viral constructcomprises the R and U5 sequences from the 5′ LTR of a lentivirus and aninactivated or self-inactivating 3′ LTR from a lentivirus. The LTRsequences may be LTR sequences from any lentivirus from any species. Forexample, they may be LTR sequences from HIV, SIV, FIV or BIV. Preferablythe LTR sequences are HIV LTR sequences.

[0069] The viral construct preferably comprises an inactivated orself-inactivating 3′ LTR. The 3′ LTR may be made self-inactivating byany method known in the art. In the preferred embodiment the U3 elementof the 3′ LTR contains a deletion of its enhancer sequence, preferablythe TATA box, Spl and NF-kappa B sites. As a result of theself-inactivating 3′ LTR, the provirus that is integrated into the hostcell genome will comprise an inactivated 5′ LTR.

[0070] Optionally, the U3 sequence from the lentiviral 5′ LTR may bereplaced with a promoter sequence in the viral construct. This mayincrease the titer of virus recovered from the packaging cell line. Anenhancer sequence may also be included. Any enhancer/promotercombination that increases expression of the viral RNA genome in thepackaging cell line may be used. In the preferred embodiment the CMVenhancer/promoter sequence is used.

[0071] In one embodiment the viral construct comprises a gene thatencodes a protein that is desirably expressed in one or more cells of atransgenic animal. Preferably the gene of interest is located betweenthe 5′ LTR and 3′ LTR sequences. Further, the gene of interest ispreferably in a functional relationship with other genetic elements, forexample transcription regulatory sequences such as promoters and/orenhancers, to regulate expression of the gene of interest in aparticular manner once the transgene is incorporated into the hostgenome. In certain embodiments, the useful transcriptional regulatorysequences are those that are highly regulated with respect to activity,both temporally and spatially.

[0072] Preferably the gene of interest is in a functional relationshipwith internal promoter/enhancer regulatory sequences. An “internal”promoter/enhancer is one that is located between the 5′ LTR and the 3′LTR sequences in the viral construct and is operably linked to the genethat is desirably expressed.

[0073] The internal promoter/enhancer may be any promoter, enhancer orpromoter/enhancer combination known to increase expression of a genewith which it is in a functional relationship. A “functionalrelationship” and “operably linked” mean, without limitation, that thegene is in the correct location and orientation with respect to thepromoter and/or enhancer that expression of the gene will be affectedwhen the promoter and/or enhancer is contacted with the appropriatemolecules.

[0074] The internal promoter/enhancer is preferably selected based onthe desired expression pattern of the gene of interest and the specificproperties of known promoters/enhancers. Thus, the internal promoter maybe a constitutive promoter. Non-limiting examples of constitutivepromoters that may be used include the promoter for ubiquitin, CMV(Karasuyama et al J. Exp. Med. 169:13 (1989), β-actin (Gunning et al.Proc. Natl. Acad. Sci. USA 84:4831-4835 (1987) and pgk (see, forexample, Adra et al. Gene 60:65-74 (1987), Singer-Sam et al. Gene32:409-417 (1984) and Dobson et al. Nucleic Acids Res. 10:2635-2637(1982)). Alternatively, the promoter may be a tissue specific promoter.Several non-limiting examples of tissue specific promoters that may beused include lck (see, for example, Garvin et al. Mol. Cell Biol.8:3058-3064 (1988) and Takadera et al. Mol. Cell Biol. 9:2173-2180(1989)), myogenin (Yee et al. Genes and Development 7:1277-1289 (1993),and thy1 (Gundersen et al. Gene 113:207-214 (1992). In addition,promoters may be selected to allow for inducible expression of thetransgene. A number of systems for inducible expression using such apromoter are known in the art, including the tetracycline responsivesystem and the lac operator-repressor system. It is also contemplatedthat a combination of promoters may be used to obtain the desiredexpression of the gene of interest. The skilled artisan will be able toselect a promoter based on the desired expression pattern of the gene inthe resulting transgenic animal.

[0075] An internal enhancer may also be present in the viral constructto increase expression of the gene of interest. For example the CMVenhancer (Karasuyama et al J. Exp. Med. 169:13 (1989) may be used incombination with the chicken β-actin promoter. Again, one of skill inthe art will be able to select the appropriate enhancer based on thedesired expression pattern.

[0076] The gene of interest is not limited in any way and includes anygene that the skilled practitioner desires to have integrated and/orexpressed in a transgenic animal. For example, the gene of interest maybe one that encodes a protein that modifies a physical characteristic ofthe transgenic animal, such as a protein that modifies size, growth, ortissue composition. In another example the gene of interest may encode aprotein of commercial value that may be harvested from the transgenicanimal.

[0077] In addition, more than one gene of interest may be placed infunctional relationship with the internal promoter. For example a geneencoding a marker protein may be placed after the primary gene ofinterest to allow for identification of cells that are expressing thedesired protein. In one embodiment a fluorescent marker protein,preferably green fluorescent protein (GFP), is incorporated into theconstruct along with the gene of interest. If a second reporter gene isincluded, an internal ribosomal entry site (IRES) sequence is alsopreferably included. The IRES sequence may facilitate the expression ofthe reporter gene

[0078] The viral construct may also contain additional genetic elements.The types of elements that may be included in the construct are notlimited in any way and will be chosen by the skilled practitioner toachieve a particular result. For example, a signal that facilitatesnuclear entry of the viral genome in the target cell may be included. Anexample of such a signal is the HIV-1 flap signal.

[0079] Further, elements may be included that facilitate thecharacterization of the provirus integration site in the genome of theanimal. For example, a tRNA amber suppressor sequence may be included inthe construct.

[0080] In addition, the construct may contain one or more geneticelements designed to enhance expression of the gene of interest. Forexample, a woodchuck hepatitis virus responsive element (WRE) may beplaced into the construct (Zufferey et al. J. Virol. 74:3668-3681(1999); Deglon et al. Hum. Gene Ther. 11:179-190 (2000)).

[0081] A chicken β-globin insulator may also be included in the viralconstruct. This element has been shown to reduce the chance of silencingthe integrated provirus in the transgenic animal due to methylation andheterochromatinization effects. In addition, the insulator may shieldthe internal enhancer, promoter and exogenous gene from positive ornegative positional effects from surrounding DNA at the integration siteon the chromosome.

[0082] Any additional genetic elements are preferably inserted 3′ of thegene of interest.

[0083] In a specific embodiment, the viral vector comprises: acytomegalovirus (CMV) enhancer/promoter sequence; the R and U5 sequencesfrom the HIV 5′ LTR; the HIV-1 flap signal; an internal enhancer; aninternal promoter; a gene of interest; the woodchuck hepatitis virusresponsive element; a tRNA amber suppressor sequence; a U3 element witha deletion of its enhancer sequence; the chicken β-globin insulator; andthe R and U5 sequences of the 3′ HIV LTR.

[0084] The viral construct is preferably cloned into a plasmid that maybe transfected into a packaging cell line. The preferred plasmidpreferably comprises sequences useful for replication of the plasmid inbacteria.

[0085] Production of Virus

[0086] Any method known in the art may be used to produce infectiousretroviral particles whose genome comprises an RNA copy of the viralconstruct described above.

[0087] Preferably, the viral construct is introduced into a packagingcell line. The packaging cell line provides the viral proteins that arerequired in trans for the packaging of the viral genomic RNA into viralparticles. The packaging cell line may be any cell line that is capableof expressing retroviral proteins. Preferred packaging cell linesinclude 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK(ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430). The mostpreferable cell line is the 293 cell line.

[0088] The packaging cell line may stably express the necessary viralproteins. Such a packaging cell line is described, for example, in U.S.Pat. No. 6,218,181. Alternatively a packaging cell line may betransiently transfected with plasmids comprising nucleic acid thatencodes the necessary viral proteins.

[0089] In one embodiment a packaging cell line that stably expresses theviral proteins required for packaging the RNA genome is transfected witha plasmid comprising the viral construct described above.

[0090] In another embodiment a packaging cell line that does not stablyexpress the necessary viral proteins is co-transfected with two or moreplasmids essentially as described in Yee et al. (Methods Cell. Biol.43A, 99-112 (1994)). One of the plasmids comprises the viral constructcomprising the transgene. The other plasmid(s) comprises nucleic acidencoding the proteins necessary to allow the cells to produce functionalvirus that is able to infect the desired host cell.

[0091] The packaging cell line may not express envelope gene products.In this case the packaging cell line will package the viral genome intoparticles that lack an envelope protein. As the envelope protein isresponsible, in part, for the host range of the viral particles, theviruses are preferably pseudotyped. Thus the packaging cell line ispreferably transfected with a plasmid comprising sequences encoding amembrane-associated protein that will permit entry of the virus into ahost cell. One of skill in the art will be able to choose theappropriate pseudotype for the host cell that is to be used. Forexample, in one embodiment the viruses are pseudotyped with thevesicular stomatitis virus envelope glycoprotein (VSVg). In anotherembodiment, a mutant ecotropic envelope protein is used, such as theecotropic envelope protein 4.17 (Powell et al. Nature Biotechnology18(12):1279-1282 (2000)). In addition to conferring a specific hostrange the pseudotype may permit the virus to be concentrated to a veryhigh titer and may enhance safety by preventing the virus from infectingundesired cell types.

[0092] In the preferred embodiment a packaging cell line that does notstably express viral proteins is transfected with the viral construct, asecond vector comprising the HIV-1 packaging vector with the env, nef,5′LTR, 3′LTR and vpu sequences deleted, and a third vector encoding anenvelope glycoprotein. Preferably the third vector encodes the VSVgenvelope glycoprotein.

[0093] The recombinant virus is then preferably purified from thepackaging cells, titered and diluted to the desired concentration.

[0094] Transgenic Animals

[0095] In order to make transgenic animals, an oocyte or one or moreembryonic cells are infected with the recombinant virus produced asdescribed above. One of skill in the art will recognize that the methodof infection and the treatment of the cell following infection willdepend upon the type of animal from which the cell is obtained. Forexample, mammalian cells are preferably implanted in a pseudopregnantfemale following infection while for the generation of transgenic birdsor fish, the virus is preferably delivered to a laid egg and thusimplantation is not required.

[0096] While early methods of making transgenic animals required thecells to be rapidly dividing, there is no such requirement in themethods of the present invention. Thus the cell may be contacted at anypoint in development. In the preferred embodiment, a zygote is contactedwith the recombinant virus.

[0097] The cells to be infected with the virus may be obtained by anymethod known in the art and appropriate for the specific species inwhich it is desired to make a transgenic animal. For example, therecovery of fertilized mouse oocytes is described in Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual. 2^(nd) ed. ColdSpring Harbor Laboratory Press, NY (1994)). A method for obtainingfertilized rat oocytes is described in Armstrong et al. (Biol. Reprod.39,511-518 (1998)).

[0098] It is not necessary that the cells be contacted afterfertilization. In one embodiment, the virus is delivered to unfertilizedova. Development may then be initialized, for example by in vitrofertilization.

[0099] Delivery of the Virus

[0100] The virus may be delivered to the cell in any way that allows thevirus to infect the cell. Preferably the virus is allowed to contact thecell membrane. Two preferred methods of delivering the virus tomammalian cells, injection and direct contact, are described below.

[0101] Injection

[0102] In a first embodiment the virus is injected into theperivitelline space between the zona pellucida and the cell membrane ofa single-cell zygote. Preferably less than 50 picoliters of viralsuspension is injected, more preferably less than 25 picoliters and evenmore preferably about 10 picoliters.

[0103] The virus is preferably present in a viral suspension and may beinjected by any method known in the art. The viral suspension ispreferably injected through a hydraulic injector. More preferably aglass micropipette is used to inject the virus. In one embodiment amicropipette is prepared by pulling borosilicate glass capillary on apipette puller. The tip is preferably opened and beveled toapproximately 10 μm. The lentiviral suspension may be loaded into themicropipette from the tip using gentle negative pressure.

[0104] In one embodiment the cell is stabilized with a holding pipettemounted on a micromanipulator, such as by gentle negative pressureagainst a fire-polished pipette, and a second micromanipulator is usedto direct the tip of a micropipette into the space between the zonapellucida and the cell membrane, where the virus is injected.

[0105] Direct Contact

[0106] In another embodiment the zona pellucida is removed from the cellto produce a denuded embryo and the cell membrane is contacted with thevirus. The zona pellucida may be removed by any method known in the art.Preferably it is removed by enzymatic treatment. For example, treatmentwith pronase may be used to remove the zona pellucida while the cellmembrane is kept intact. Alternatively, the cell may be placed in mediaat pH at which the zona pellucida dissolves while the cell membraneremains intact. For example the cell may be incubated in an acidicTyrode's solution at room temperature for several minutes. Once the zonapellucida is removed, any method that allows for the virus to contactthe cell membrane may be used. Preferably, the cell is incubated in asolution containing the virus. Even more preferably, the solution ismedia that facilitates survival of the cell.

[0107] In this embodiment, the cells are preferably contacted with thevirus in culture plates. The virus may be suspended in media and addedto the wells of a multi-well culture plate. The cells may then be platedin the individual wells. The media containing the virus may be addedprior to the plating of the cells or after the cells have been plated.Preferably individual cells are incubated in approximately 10 μl ofmedia. However, any amount of media may be used as long as anappropriate concentration of virus in the media is maintained such thatinfection of the host cell occurs.

[0108] The cells are preferably incubated with the virus for asufficient amount of time to allow the virus to infect the cells.Preferably the cells are incubated with virus for at least 1 hour, morepreferably at least 5 hours and even more preferably at least 10 hours.

[0109] Both the injection and direct contact embodiments mayadvantageously be scaled up to allow high throughput transgenesis.Because of the relative simplicity of the injection technique, it ispossible to inject many embryos rapidly. For example, it is possible toinject more than 200 fertilized oocytes in less than one hour. Withregard to the direct contact embodiment, any number of embryos may beincubated in the viral suspension simultaneously. This may beaccomplished, for example, by plating the desired number of single-cellzygotes in multi-well tissue culture plates containing the virussuspended in media appropriate for the survival and growth of the cells.

[0110] In both embodiments, any concentration of virus that issufficient to infect the cell may be used. Preferably the concentrationis at least 1 pfu/μl, more preferably at least 10 pfu/μl, even morepreferably at least 400 pfu/μl and even more preferably at least 1×10⁴pfu/μl.

[0111] Following infection with the virus, the cells are preferablyimplanted in an animal. More preferably cells infected with the virusare implanted in pseudo-pregnant animals of the same species from whichthe infected cells were obtained. Methods of creating pseudo-pregnancyin animals and implanting embryos are well known in the art and aredescribed, for example, in Hogan et al. (Manipulating the Mouse Embryo:A Laboratory Manual. 2^(nd) ed. Cold Spring Harbor Laboratory Press, NY(1994)).

[0112] In the preferred embodiment early stage embryos (approximately0-2.5 days p.c.) still with an intact zona pellucida are transferred tothe oviduct of timed pseudopregnant female (preferably 0.5 days p.c.),while embryos that have reached the blastocyst stage are transferred tothe uterus of timed pseudopregnant females (preferably 2.5 days p.c.).Denuded embryos are preferably cultured in vitro until they reach themorula or blastocyst stage (48 to 72 hours in culture), and are thenimplanted into appropriately timed pseudopregnant females.

[0113] The embryos and resulting animals may be analyzed, for examplefor integration of the transgene, the number of copies of the transgenethat integrated, the location of the integration, the ability totransmit the transgene to progeny and expression of the transgene. Suchanalysis may be carried out at any time and may be carried out by anymethods known in the art. Standard techniques are described, forexample, in Hogan et al. (supra).

[0114] The methods of infecting cells disclosed above do not depend uponspecies-specific characteristics of the cells. As a result, they arereadily extended to all mammalian species.

[0115] Initial experiments with mice indicate that of those animals thatdevelop to full term, 80-90% carried at least one copy of the transgeneand that, of these, approximately 85% express the gene of interest. Ofthe transgenic animals about 25% carry only 1 or 2 copies of thetransgene. The highest number of proviral insertions observed was about30. Of the animals that carried only 1 or 2 copies of the transgene,about 80% expressed the gene of interest.

[0116] As discussed above, the modified retrovirus can be pseudotyped toconfer upon it a broad host range. One of skill in the art would also beaware of appropriate internal promoters to achieve the desiredexpression of a gene of interest in a particular animal species. Thus,one of skill in the art will be able to modify the method of infectingcells to create transgenic animals of any species.

[0117] For example, transgenic birds are created by delivering amodified retrovirus, as described above, to the primordial germ cells ofearly stage avian embryos. In one embodiment, freshly laid eggs areobtained and placed in a temperature controlled, humidified incubator.Preferably, the embryonic blastodisc in the egg is gradually rotated tolie on top of the yolk. This may be accomplished by any method known inthe art, such as by gently rocking the egg regularly, preferably every15 minutes. Approximately 36 hours later, the modified retrovirus isdelivered into the space between the embryonic disk and theperivitelline membrane. Preferably about 50 nL of viral solution isdelivered, more preferably about 100 nL of viral solution is delivered,and even more preferably about 200 nL of viral solution is delivered.The viral solution may be delivered by any method known in the art fordelivering compositions to the inside of an egg. In the preferredembodiment a window is opened in the shell, the viral solution isinjected through the window and the shell window is closed. The eggs arepreferably incubated until hatching. The eggs will hatch afterapproximately 20 days, depending upon the particular avian species fromwhich they are obtained. Hatched chicks are preferably raised to sexualmaturity and mated. The transgenic offspring of the founder animals maybe identified by any method known in the art, such as Southern blot, PCRand expression analysis.

[0118] In another embodiment, transgenic fish are created by deliveringthe modified retrovirus, described above, to single-cell fish embryos.Fertilized fish eggs are collected by any method known in the art. Themodified retrovirus is then preferably delivered to the space betweenthe chorion and the cell membrane. This may be accomplished, forexample, by loading the modified retrovirus in solution into a glasspipette. The pipette may then be used to pierce the chorion membrane anddeliver the viral suspension. Preferably about 50 nL of viral solutionis delivered, more preferably about 100 nL of viral solution isdelivered, and even more preferably about 200 nL of viral solution isdelivered. Injected embryos are preferably returned to atemperature-controlled water tank and allowed to mature. At sexualmaturity the founder fish are preferably mated and their progenyanalyzed for the presence of the transgene by any method known in theart.

[0119] As mentioned above, the methods of the present invention willalso prove useful in techniques for identifying genes that are involvedin specific biological processes, such as gene trap assays andlarge-scale mutagenesis screens.

[0120] Gene trap experiments allow the identification and cloning of agene that is expressed in a particular tissue or cell type, and/or at aparticular time, based solely on its pattern of expression. Genetrapping relies on the capture of the splicing donor of an mRNA byectopically inserting a downstream splice acceptor, in this case,carried within an integrated provirus. Gene trapping has beensuccessfully used in several model systems, including the fruit flyDrosophila, mammalian cells in culture, and mouse ES cells (which havethe advantage of being able to be used to derive mice afterwards forfurther analysis). Gene trapping in cell culture has the advantage ofbeing fast and inexpensive, but is limited by the inability of the cellsto differentiate into specific cell types. Thus, gene trappingexperiments in mammalian cell lines in culture usually yield onlyhousekeeping genes expressed non-specifically in any mammalian cell, orcell-specific genes that are only expressed by the particular cell linein vitro. Because cell lines often show incomplete degrees ofdifferentiation, the complement of tissue-specific genes expressed bythese cells is limited. Furthermore, there are many tissues for whichrepresentative cell lines do not exist.

[0121] The use of the above-described recombinant lentiviral vectors forthe purposes of gene trapping is facilitated by the self-inactivatingmutation in the U3 enhancer element of the 3′ LTR. The lack oftranscriptional activity from the integrated 5′ LTR ensures that anytranscription of a reporter element in the provirus is driven byupstream regulatory sequences to the insertion that have been “trapped,”rather than from the viral promoter itself.

[0122] Thus, one embodiment of the present invention concerns a methodof identifying genes that are expressed in a particular tissue and/or ata particular time during the development of an organism. Aself-inactivating viral construct is made that preferably comprises asplice acceptor sequence and a sequence encoding a reporter gene.Modified retroviral particles are made using the viral construct asdescribed above and used to infect embryonic cells. Tissues from thefounder animal or its progeny are analyzed for the presence of thereporter to determine the temporal and/or spatial pattern of expression.Messenger RNA is collected from the tissues of the animals that expressthe reporter protein in the time and place of interest. The “trapped”gene may then be identified by any method known in the art. Preferably,oligonucleotides that are complementary to the reporter gene may thenused in a reverse transcription reaction to produce a cDNA that containsthe sequences of the trapped gene that flank the provirus. The cDNA maythen be cloned into a plasmid from which the gene may be identified bynucleotide sequencing.

[0123] Gene trap experiments are well known in the art and the skilledartisan would be able to choose the reporter gene, splice acceptorsequence and any other genetic elements that would be useful to includein the viral construct based on the specific analysis that they haveundertaken. In addition, by modifying the viral constructs, thetechnique can be used to trap promoter or enhancer sequences. Forpromoter trap experiments, the reporter gene lacks any transcriptionalregulatory elements, and is only expressed when the virus integratesnext to an active promoter. For the enhancer trap, the reporter gene ispositioned downstream of a minimal promoter that lacks transcriptionalactivity, and is only expressed when the virus integrates next to anactive enhancer.

[0124] Another important paradigm by which biologists study genefunction is to disrupt the function of an endogenous gene and, from themutant phenotype that results, deduce the normal role of that gene inthe organism. One way of isolating genes that are important in aparticular biological process under study is to perform large-scalemutagenesis to generate animals that are phenotypically mutant in thatprocess and then to isolate the gene that is disrupted in the mutantanimal and that is thus responsible for the mutant phenotype. In mostsuch experiments, either radiation or chemicals have been used to inducedeletions or nonsense mutations. However, the genes carrying mutationsinduced by radiation or chemicals are difficult to isolate because nohandle is available with which to clone the gene. Rather, thesemutations must be identified by positional cloning, a slow andpainstaking process in which the mutation of interest is systematicallymapped relative to known genetic markers in the genome, with the goal ofgradually narrowing down and pinpointing the locus of the mutated gene.

[0125] In contrast, a powerful technique that has been used successfullyin the fruit fly Drosophila melanogaster is that of insertionalmutagenesis, in which genes are disrupted when an exogenous piece of DNAis inserted within the coding sequence of the gene. The great advantageof insertional mutagenesis is that, because the sequence of theexogenous disrupting DNA is known, one can directly clone out that pieceof DNA and the flanking sequence that corresponds to the gene ofinterest that has been disrupted. Thus, in contrast to traditionalpositional cloning strategies used in chemical mutagenesis which maytake up to 3 years after the isolation of the mutant, identification andcharacterization of the mutated gene of interest in an insertionalmutagenesis strategy is reduced to just a week or so. The mainlimitation to the application of insertional mutagenesis in organismsother than the fruit fly is the lack of a DNA element, such as thetransposon used in Drosophila, that is able to stably integrate and markits position in the genome at the germline or one-cell embryo stage.

[0126] The lentiviral vectors described above can be effective tools forlarge-scale mutagenesis to identify genes involved in specificbiological processes. The modified lentiviruses of the present inventionare easily delivered to the germline, and pseudotyping of the viruseswith an envelope glycoprotein, such as VSVg, allows the concentration ofthe virus to extremely high titers. Thus in one embodiment mutagenesisis achieved by delivery of the modified retrovirus to the cell membraneof embryonic cells.

[0127] The ability of transgenic animals made by the methods of thepresent invention to express a gene of interest at high levels suggeststhat the integrated proviruses are not silenced by methylation. Previousmutagenesis screens using MoLV-based retroviruses have been limited bythe observation that, in addition to the provirus, flanking genomicsequences are frequently found to be methylated and inactivated. Thismethylation complicates the analysis because it becomes difficult todistinguish whether the mutant phenotype is due to the disruption of thegene into which the provirus has inserted, or due to the inactivation ofany one of several surrounding genes by methylation.

[0128] By delivering modified lentiviruses to embryos according to themethods of the present invention, insertional mutagenesis strategies canbe applied to any animal species, including model genetic organisms suchas Xenopus, zebrafish, mouse, rat, and zebra finch. Early-stage embryos,consisting of several cells, will preferably be targeted because theresulting mosaicism increases the number of unique mutagenic events thatcan be screened.

[0129] The modified lentiviruses integrate randomly into the genome ofthe target zygotes, including that of the germ cells. Thus, someproportion will disrupt coding sequences. The embryos are preferablyraised to sexual maturity, mated, and the progeny are screened formutant phenotypes of interest. Once a mutant is identified, selectivebreeding using standard methods is preferably used to isolate theparticular insertion(s) responsible for the phenotype.

[0130] Once a mutant line is established, the mutated locus ispreferably identified using the provirus as a handle for cloning. In oneembodiment, an origin of replication and antibiotic resistance gene isincluded in the viral construct. In this embodiment, genomic DNA fromthe mutant is preferably isolated, randomly cleaved with an appropriaterestriction enzyme, and the linear fragments circularized by ligation.The ligation mixture is then transformed into bacterial cells and platedon antibiotic plates. The plasmid DNA from any colonies that arise isisolated and preferably used as a template for inverse PCR withoppositely oriented, adjacent primers complementary to sequences in theprovirus. The amplified DNA molecule(s) is then sequenced to acquire theflanking regions to the integration site, corresponding to the gene(s)mutated.

[0131] In another embodiment, inclusion of the tRNA amber suppressorsequence in the provirus allows for the rapid generation of genomiclibraries containing the flanking regions of the integration loci,representing the disrupted gene(s). Once these flanking regions aresequenced, they can be compared against the genomic sequence databasefor that animal to determine candidate gene(s) of interest.

[0132] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

[0133] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLE 1

[0134] Transgenic mice were generated that expressed a heterologousprotein, green fluorescent protein (GFP). GFP expression was controlledby manipulating the genetic elements in the viral construct employed tocreate the transgenic mice. For example, a viral construct, FUGW,comprising a ubiquitous promoter was used to produce transgenic micethat expressed GFP in every cell. Inclusion of a nuclear localizationsignal produced transgenic mice that had GFP localized in the nucleus oftheir cells. A viral construct with a lymphocyte specific promoterproduced mice that expressed GFP in lymphocytes, while a viral constructwith a muscle specific promoter produced mice that specificallyexpressed GFP in muscle cells.

[0135] A. Viral Constructs

[0136] A viral construct according to the present invention was createdusing the HR'CS-G plasmid (Miyoshi H, Blomer U, Takahashi M, Gage F H,Verma I M. J Virol. 72(10):8150-7 (1998)). This plasmid is based on theHIV-1 HXB2 proviral DNA (see U.S. Pat. No. 6,013,516).

[0137] 1. Generation of a Vector Expressing GFP from a UbiquituousPromoter, FUGW

[0138] The HIV-1 flap sequence (SEQ ID NO: 1) was inserted into theHR'CS-G vector. A 147 base pair sequence containing the flap region(Zennou, V., Petit, C., Guetard, D., Nerhbass, U., Montagnier, L.,Chameau, P. Cell. 101(2),173-185 (2000)) was PCR amplified from aplasmid encoding the genome of HIV NLA4.3. The 5′ PCR primer encodedBglII and PacI sites. The 3′ PCR primer contained the BamHI site. Theresulting PCR product was digested with BglII and BamHI enzymes andinserted into the BamHI site of the HR'CS-G vector. The resultingplasmid was called Hflap, representing the Hflap sequence that theplasmid contains.

[0139] The ubiquitin promoter (SEQ ID NO: 2) was then inserted into theHflap plasmid. The 1.2 Kb sequence encoding the human polyubiquitin Cpromoter was excised with BglII and BamHI enzymes and inserted into theBamHI site of Hflap. The resulting plasmid was called HflapUbi,representing the HflapUbi sequence that the plasmid contains.

[0140] A multi-cloning site was then inserted into HflapUbi. Twooligonucleotides were designed that encoded the following restrictionsites: BamHI HpaI XhoI AscI EcoRI BglII. The oligos were hybridized andinserted into the BamHl site of HflapUbi. The resulting plasmid wascalled FUMCS.

[0141] A nucleic acid sequence encoding GFP (SEQ ID NO: 3) was theninserted into HflapUbi. The 700 base pair sequence of GFP was digestedwith BamHI and XhoI and inserted into the XhoI site of HflapUbi,generating HflapUbiG which represents the HflapUbiG sequence that theplasmid contains. The resulting plasmid was called FUG.

[0142] The woodchuck hepatitis virus regulator element (WRE; SEQ ID NO:4) was then inserted into HflapUbiG. The 500 bp sequence of WRE(Zufferey, R., Donello, J. E., Trono, D., Hope T. J. (1999). J Virol.73(4), 2886-92) was excised with SalI and XhoI and inserted into theXhoI site of HflapUbiG generating a plasmid containing the HflapUbiGWREsequence (SEQ ID NO: 8). The resulting plasmid was called FUGW (SEQ IDNO: 9). A map of the FUGW viral vector is presented in FIG. 1A.

[0143] 2. Generation of a Vector Expressing Nuclear-Localized GFP from aUbiquituous Promoter, FUH2BGW

[0144] In order to get specific nuclear localization of GFP, the histone2B-GFP fusion sequence H2BGFP was cloned into FUGW. The histone 2B-GFPsequence (SEQ ID NO: 5) was digested with SalI and NotI. Both sites wereblunted with T4 DNA polymerase and inserted into the HpaI site of FUMCS.The resulting plasmid was called FUH2BGW.

[0145] 3. Generation of a Vector Expressing GFP from aLymphocyte-Specific Promoter, FlckGW

[0146] To achieve lymphocyte specific GFP expression the murine lckpromoter (SEQ ID NO: 6) was cloned into the FUGW vector. The ubiquitinpromoter from FUGW was removed by excising with PacI and BamHl. The PacIsite was blunted using T4 DNA polymerase and the lck promoter wasexcised with SpeI and BamHI. The Spel site was blunted using T4 DNApolymerase. The lck promoter was the inserted into the PacI and BamHIsites of FUGW. The resulting plasmid was called FlckGW.

[0147] 4. Generation of a Vector Expressing GFP from a Muscle-SpecificPromoter, FmyoH2BGW

[0148] To achieve specific expression of GFP in the muscle of transgenicmice the myogenin promoter (SEQ ID NO: 7) was cloned into the FUGWconstruct. The mouse myogenin promoter was PCR amplified from a mousegenomic BAC. The 5′ PCR primer encoded a PacI site, and the 3′ PCRprimer contained an XbaI site. The PCR product was digested with PacIand XbaI. The ubiquitin promoter was removed from the FUH2BGW vector bycutting with PacI and XbaI. The PacI-BamHI digested PCR product encodingthe myogenin promoter was inserted into the PacI and XbaI sites of theFUH2BGW vector.

[0149] The constructs described above were then used to preparerecombinant lentivirus. Briefly, replication-incompetent viral vectors,based on the human immunodeficiency virus-1 (HIV-1), were pseudotypedwith the vesicular stomatitis virus envelope glycoprotein (VSVg),permitting the virus to be concentrated to very high titers andconferring upon the virus a broad host range. Pseudotyped lentiviruseswere produced essentially as described in detail in Yee, J. K.,Friedmann, T. & Burns, J. C. (1994). Methods Cell Biol. 43, 99-112;Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K.(1993). Proc. Natl. Acad. Sci. USA. 90, 8033-8037; and Yee, J. K.,Miyanohara, A., LaPorte, P., Bouic, K., Burns, J. C., and Friedmann, T.(1994). Proc. Natl. Acad. Sci. USA. 91, 9564-9568. Briefly, humanfibroblasts 293 cells were transfected with calcium phosphate/DNAcoprecipitates of the following plasmids, as described in Gorman, C.,Padmanabhan, R. and Howard, B. H. (1983). Science. 221, 551-553:

[0150] The viral transfer vector described above with self-inactivatingLTR;

[0151] the HIV-1 packaging vector Δ8.9 (Zufferey, R., Nagy, D., Mandel,R. J., Naldini, L., and Trono, D. (1997). Nat. Biotechnol. 15(9),871-875; Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R.,Gage, F. H., Verman, I. M., and Trono, D. (1996). Science. 272(5259),263-67) with env, vpr, vpu, vif, nef, 5′LTR, 3′LTR, and ψ sequencesdeleted; and the VSVg envelope glycoprotein.

[0152] Viral supernatant was harvested 60 hours post-transfection,subjected to low-speed centrifugation to remove cell debris, filteredthrough a 0.45 μm nitrocellulose membrane, spun at 25,000 rpm for 1.5hours to concentrate, and resuspended in a small volume (one hundredthto one thousandth of the original volume) of phosphate-buffered saline(PBS), pH 7.4. The titer of the viral concentrate was approximately1×10⁶ pfu/μl as determined in 293 human fibroblasts measured by thenumber of GFP-positive cells. The viral suspension was stored frozen at−80° C.

[0153] B. Production of Transgenic Mice and Rats

[0154] The lentivirus was used to produce transgenic mice and rats.

[0155] 1. Superovulation and Embryo Collection

[0156] Female mice and rats were superovulated with a combination ofpregnant mare's serum (PMS) and human chorionic gonadotropin (hCG) asdescribed in Hogan, B., Beddington, R., Costantini, F., and Lacy, E.(1994). Manipulating the Mouse Embryo: A Laboratory Manual. Cold SpringHarbor Laboratory Press. “Superovulation” refers to administeringgonadotropins to female mammals prior to mating to increase the numberof eggs that are ovulated. Prepubescent female mice (approximately 25days of age and weighing between 12.5 and 14 grams) were injectedintraperitoneally with 5 IU of PMS (Sigma G 4527, 25 IU/ml in 0.9% NaCl)between 1 and 3 p.m. on day −2, followed by 5 IU of HCG (Sigma C 8554,25 U/ml in 0.9% NaCl) 48 hours later on day 0. Prepubescent female ratsbetween 28-30 days of age and weighing between 70 and 80 grams wereinjected intraperitonally with 25 IU of PMS between 1 and 3 p.m. on day−2, followed by 5 IU of HCG 48 hours later on day 0. For both rats andmice, hormone-treated females were caged overnight with fertile males(2-3 months of age) to mate. On the morning of day 1, females werechecked for copulation plugs.

[0157] Female mice were sacrificed for embryo collection around 10 a.m.on the morning of day 1, while female rats were sacrificed forcollection around 1 p.m. on the afternoon of day 1. Embryos werecollected from mice and rats essentially following the proceduredescribed in Hogan, B., Beddington, R., Costantini, F., and Lacy, E.(1994) Manipulating the Mouse Embryo: A Laboratory Manual. Cold SpringHarbor Laboratory Press. Briefly, animals were sacrificed by CO₂inhalation, and the oviducts were excised and transferred to a dishcontaining M2 medium at room temperature. Newly fertilized embryos,enclosed by cumulus mass cells, were released from the swollen ampullae(the upper portion of the oviduct) by gently tugging and opening thewalls of the ampullae with fine forceps. The embryos were thentransferred to a dish containing a hyaluronidase solution (Sigma H 3884,300 μg/ml in M2 medium), which enzymatically digested the cumulus cells,thus releasing the embryos. When the cumulus cells were shed, theembryos were transferred to fresh M2 medium to wash off thehyaluronidase solution and preserve the viability of the embryos. Inrats, the cumulus cells were found to adhere tenaciously to the surfaceof some embryos and were difficult to remove completely. Thus in somecases the subsequent experimental manipulations with the zygotes werecarried out with some of the cumulus cells still adhering. This did notseem to affect the outcome. The embryos were then transferred tomicrodrops of M16 medium under mineral oil and cultured in a humidified37° C. incubator under 5% CO₂ until needed.

[0158] 2. Delivery of Lentiviruses to Single-Cell Embryos

[0159] Lentiviruses were delivered to the fertilized oocytes on the sameday of collection, targeting only single-cell zygotes to minimizemosaicism. Infection with lentivirus derived from the FUGW viralconstruct will lead to integration of the provirus locus diagrammed inFIG. 1B. Two different methods were used to deliver the lentiviruses tothe embryos:

[0160] a. Microinjection of Lentiviruses into the Perivitelline Space ofSingle-Cell Embryos

[0161] Micropipettes were prepared by pulling borosilicate glasscapillaries (1 mm O.D., 0.7 mm I.D.) on a Sutter Instruments pipettepuller. The tip was cut at an angle to approximately 10 μm with a razorblade. The micropipette was then inserted into the pipette holder of aCellTram hydraulic injector (Eppendorf). The lentiviral concentrateprepared above was pipetted up and down to release any large aggregatesof cellular debris. The virus was centrifuged at low speed in a tabletopmicrofuge (1000 rpm for 1 min.), and removed from the top. The viralsuspension was then loaded into the micropipette from the tip usinggentle negative pressure from the CellTram.

[0162] One-cell embryos were transferred to a microdrop of M2 medium ona slide and covered with mineral oil to maintain the osmolarity. Theslide was mounted on the stage of an inverted light microscope, and theinjection procedure was monitored under 400× magnification. Embryos wereheld in place against a fire-polished pipette using gentle negativepressure. The pipette holder with the virus was loaded onto amicromanipulator (Leitz). Using the micromanipulator to guide thepipette, the tip was pushed through the zona pellucida into the regionbetween the zona pellucida and the oocyte cell membrane. Using gentlepositive pressure, approximately 10 nanoliters of the viral concentratewas delivered into the perivitelline space. The micropipette was thenwithdrawn from the zygote. After the injection, the embryos were sortedand those that were lysed, abnormal, or at the 2-cell stage werediscarded. The remaining embryos were transferred to M16 microdropsunder oil and cultured in a 37° C. incubator under 5% CO₂ untilimplantation.

[0163] b. Co-Incubation of Denuded Single-Cell Embryos with Lentiviruses

[0164] The zona pellucida of the fertilized oocytes was removed byincubation in either an acidic Tyrode's solution (Hogan, B., Beddington,R., Costantini, F., and Lacy, E. (1994). Manipulating the Mouse Embryo:A Laboratory Manual. Cold Spring Harbor Laboratory Press) or a 0.5%pronase solution in M2 medium at 37° C. in a humidified 5% CO₂ incubatorfor several minutes. When the zonae appeared to be dissolved, embryoswere washed in excess M2 medium and then transferred into 10 μlmicrodrops of viral suspension under mineral oil. Embryos were culturedindividually in separate microdrops to prevent them from adhering to oneanother. The viral suspension was diluted to various concentrations toroughly control the average number of proviral integrations expected pertransgenic genome. Virus in the microdrops was diluted to 2×10⁴ pfu/μl,400 pfu/μl, and 8 pfu/μl. Zygotes were incubated in the viral suspensionfor at least 4-6 hours before implantation to allow viral entry into thecell.

[0165] 3. Transfer of Embryos into Recipient Females

[0166] Timed pseudopregnant females to host the treated embryos wereprepared by mating sexually mature females in estrus to vasectomized,mature males the night before the intended day of implantation.Appropriate females were selected from a colony of 30-40 females bytaking vaginal smears and examining them for the cell types typical ofthe estrus phase. Males were vasectomized by tying off the vas deferensat two separate locations, approximately 5-6 mm apart, then cauterizingthe intervening segment to sever the tube. Males were vasectomized atleast 2 weeks prior to the mating to ensure that all remaining sperm inthe genital tract were dead at the time of mating.

[0167] Embryos infected with lentivirus were transferred into hostfemales as soon as possible to achieve maximum rates of implantation.Early-stage embryos (0-2.5 days p.c.) with an intact zona pellucida weretransferred to the oviduct of timed pseudopregnant females (0.5 daysp.c.), while embryos that had reached the morula or blastocyst stagewere transferred to the uterus of timed pseudopregnant females (2.5 daysp.c.). In general, no more than 30 embryos were transferred bilaterallyinto the uterus. These procedures were carried out essentially asdescribed in (Hogan, B., Beddington, R., Costantini, F., and Lacy, E.(1994). Manipulating the Mouse Embryo: A Laboratory Manual. Cold SpringHarbor Laboratory Press). Pregnancy and delivery of the transgeniclitter was as usual.

[0168] C. Analysis of Transgenic Animals

[0169] Animals in the resulting litters were analyzed for the presenceof the transgene and the number of insertions of the transgene bystandard Southern blot analysis (Sambrook, J., Fritsch, E. F., Maniatis,T. (1989). Molecular Cloning: A Laboratory Manuel. Cold Spring Harborlaboratory Press.), cutting with PstI or BamHI and hybridizing against aGFP+WRE probe. For constitutive promoters, expression of GFP wasdetermined by directly viewing the skin of the animals under aconventional epifluorescence microscope. Some transgenic animals thatwere scored as negative for expression were actually expressing thetransgene at levels below that of detection by visual inspection with afluorescent microscope. In such cases, western blot analysis revelaedthat animals in which GFP fluorescence was not detected by visualinspection did express the GFP protein in some tissues. Similarly,immunocytochemistry proved to be a more sensitive assay for determiningexpression. For the tissue-specific promoters, some proportion of thetransgenic litter was sacrificed during development at embryonic stages,and the translucent embryos were checked for spatially regulated GFPexpression under a fluorescent microscope. Expression results wereconfirmed by histology. To test the ability of the founder animals totransmit the transgene to their progeny, animals positive by Southernanalysis were outcrossed to wild-type animals, and their progeny scoredfor transgenesis and expression as described above.

[0170] D. Results

[0171] In one set of experiments one-cell mouse and rat embryos wereinjected in the perivitelline space with recombinant lentivirus asdescribed above. In the first experiment, 17 founder mice developed toterm from 78 implanted embryos. Of these, 11 of the 17 foundersexpressed the transgene as determined by directly viewing the animalsunder an epifluoresence microscope. Further, 11 of 15 (two mice diedprior to analysis), or approximately 73%, were found to carry thetransgene by Southern blot analysis. The average number of insertions inthe transgenic mice was 6.1. Several of the animals carried as few as 2insertions. These results are presented in FIG. 2.

[0172] In a second experiment, 56 founder mice developed to term from119 implanted embryos. Of these 45, or about 80%, were found to expressthe transgene. Thus, in the two experiments 58 out of 73 founder mice,approximately 79.5%, carried the transgene. FIG. 3 shows the Southernblot analysis of proviral transgene insertions in these founder mice.

[0173] All GFP-positive mice carried an integrated provirus, and allanimals with two or more copies of the provirus expressed the transgeneat levels detectable by direct viewing of GFP fluorescence. Theintensity of GFP fluorescence correlated positively with copy number, asestimated qualitatively. All major tissues and organs, including skin,bone, skeletal muscle, cardiac muscle, lung, liver, thymus, spleen,stomach, intestine, kidney, brain, retina and gonads were GFP positive.

[0174] In a third experiment, five rats developed to term from embryosinjected with lentivirus created from the FUGW construct. Two of thefive rats were found to express the transgene as determined bybrightfield and fluorescent images of the paws of the newborn rats (FIG.4A). Pup R4 expresses GFP in the paw, as well as in all other tissuesand organs that were examined (FIG. 4A). FIG. 4B shows the Southern blotanalysis of the proviral insertions in these founder rats and indicatesthat pup R4 carries 4 copies of the proviral insert.

[0175] In a continuation of this experiment, out of 22 founder rat pupsborn from 130 implanted embryos, 13 (59.1%) carried one or more proviralinsertions as determined by Southern blot analysis and 9 (40.9%)expressed GFP at levels detectable by directly viewing the skin under afluorescent microscope. GFP positive founders were crossed to wild-typeanimals, and F1 progeny rats carrying as few as one copy of the provirusexpressed GFP, as determined by direct viewing with a fluorescentmicroscope, indicating that the GFP-expressing transgene is not silencedby transmission through the germline.

[0176] In another set of experiments denuded mouse embryos wereincubated in decreasing concentrations of recombinant lentivirus. Arough correlation was seen between the titer of virus in which embryoswere incubated and the number of proviral insertions. At a 1:50 dilutionfrom a stock of 1×10⁶ pfu/μl 5 founder mice that reached term (from 29implanted embryos) were found to be transgenic. All of these animalscarried at least 6 proviral insertions. The average number of insertionswas 7.2. Of these, 4, or 80%, were found to express the transgene. At a1:250 dilution five out of 7 founder mice that reached term (from 18implanted embryos) were found to be transgenic and express thetransgene. In these mice the average number of insertions was 3.8, withtwo of the animals carrying only one or two copies of the transgene.Finally, at a dilution of 1:1250 only one of the 8 founders (from 40implanted embryos) was found to be transgenic, with a single insertion.This founder also expressed the transgene. FIG. 5 shows the Southernblot analysis of proviral transgene insertions in these founder mice.FIG. 6 shows GFP expression in one of the founder mice. A second trialwith a 1:250 dilution gave comparable results. Eight of 11 founder mice(from 59 implanted embryos) were transgenic, with seven expressing thetransgene. The transgenic mice had an average of 2.6 insertions.

[0177] Following outcrossing to wild-type animals, progeny were analyzedfor proviral transgene insertions by Southern blot (FIG. 7) and for GFPexpression by viewing under an epifluoresence microscope (FIG. 8). Ascan be seen in Table 1, founder mice were able to transmit the transgeneto their progeny. In Table 1, “PV” represents founder transgenicsgenerated by injection of the lentivirus into the perivitelline space ofone-cell embryos while “Co-inc” represents founder transgenics generatedby co-incubation of the denuded embryos with lentivirus. TABLE 1 No.insertions in No. Founder founder No. progeny expressing PV.13 6 7 6/7PV.2 2 4 2/4 PV.10 2 7 1/7 PV.17 10 10 10/10 Co-inc.18 12 12  9/12Co-inc.2 0 9 0/9

[0178] Ubiquitous GFP expression similar to that of the founder animalswas observed in transgenic F1 progeny, indicating that the provirus wasnot inactivated through one round of gametogenesis and development. Allanimals carrying two or more insertions of the FUGW provirus expressedGFP at levels detectable by direct fluorescence. However, amongtransgenic lines carrying one proviral insertion, approximately halfexpressed the transgene at levels detectable by direct fluorescence. Inone single insertion line in which GFP expression was not observed bydirect viewing, GFP was detectable by Western blot analysis in sometissues (brain, testes) but not in others (heart, lung, liver, kidney,spleen).

[0179] In a further experiment, single-cell mouse zygotes were injectedin the perivitelline space with recombinant lentivirus derived from theFMH2BGW viral construct described above. This construct comprises ahistone2B-GFP fusion gene under the control of the myogenin promoter.The histone2B-GFP reporter was used to concentrate the fluorescence inthe nuclei, making the signal more intense. Zygotes were implanted inpseudopregnant female mice and then recovered from the uterus atembryonic day 11.5. As can be seen in FIG. 9, all six founder-mice weretransgenic as determined by Southern blot analysis of proviral transgeneinsertions. Of these, two animals were positive for tissue-specific GFPexpression at embryonic day 11.5 (FIG. 9).

[0180]FIG. 10 shows that at embryonic day 11.5, GFP expression islocalized to the somites and can be seen in the emerging muscles in thelimb buds, eye and jaw. This expression pattern is consistent withmyogenin expression at this stage of development. FIG. 11 shows theresults of immunofluoresence studies of sections of an embryonic day11.5 embryo carrying the myogenin promoter driving GFP. Specificstaining of somite tissues can be seen (FIG. 11). FIGS. 12 and 13 showfurther immunofluorescence studies of cross-sections of an E11.5-embryocarrying a myogenin promoter driving GFP.

[0181] Fifteen-day old animals, derived from FMH2BGW-infected zygotesshowed GFP fluorescence in the nuclei of skeletal muscle in the tongue,limbs, chest and jaw, but not in cardiac or smooth muscle or othernon-muscle tissues examined, reflecting the known specificity ofmyogenin expression. F1 progeny from three independent foundersexpressed histone2B-GFP exclusively in the skeletal muscle lineage.Furthermore, progeny carrying as few as one FMH2BGW proviral insertionexpressed histone2B-GFP in the appropriate tissue types at high levelsdetectable by direct viewing with a fluorescent microscope.

[0182] In a further experiment, a viral vector containing GFP driven bythe T-lymphocyte promoter lck, FlckGW was delivered to the perivitellinespace by injection as described above. The resulting transgenic miceexpressed GFP exclusively in the thymus.

EXAMPLE 2

[0183] Transgenic birds, such as chicken or quail, may be made by themethods of the present invention.

[0184] Freshly laid chicken eggs (day 0) are placed in atemperature-controlled, humidified incubator at 38° C. The embryonicblastodisc is gradually rotated to lie on top of the yolk by gentlyrocking the eggs in the incubator every 15 minutes. A window is openedin the shell and the blastodisc is visualized in freshly laid eggs (0hours post-laying) or stage X embryos (36 hours post-laying).VSV-pseudotyped lentiviral particles in solution are loaded into a glasscapillary micropipette. To maximize the chances of targeting primordialgerm cells, virus is injected in the anterior regions of the O hourembryos and in the gonadal anlage of the 36 hours embryos. Approximately200 nL of viral solution are delivered into the space between theperivitelline membrane and the embryonic disk with the aid of ahydraulic injector. The shell window is then closed with a porous tapeto allow gas exchange between the embryo and the incubator atmosphere.The embryos are then incubated without rocking. The eggs will hatchafter approximately 20 days of incubation time. Hatched chicks areraised to sexual maturity and then mated. The eggs laid by the matedfemales are raised to hatching and the resulting transgenic chicks areidentified, such as by Southern blot, PCR or expression analysis.

EXAMPLE 3

[0185] Transgenic zebra finch were made by the methods of the presentinvention.

[0186] Freshly laid zebra finch eggs (day 0) were placed in atemperature-controlled, humidified incubator at 38° C. The embryonicblastodisc was gradually rotated to lie on top of the yolk by gentlyrocking the eggs in the incubator every 15 minutes. A window was openedin the shell and the blastodisc was visualized. VSV-pseudotypedlentiviral particles in solution were loaded into a glass capillarymicropipette. The lentivirus was derived from the FUH2BGW viralconstruct described above. To maximize the chances of targetingprimordial germ cells, virus was injected in the anterior regions of 0hour embryos and in the gonadal anlage of 36 hour embryos. Approximately200 nL of viral solution are delivered into the space between theperivitelline membrane and the embryonic disk with the aid of ahydraulic injector.

[0187] The shell window was closed with a porous tape to allow gasexchange between the embryo and the incubator atmosphere. The embryoswere then incubated without rocking. FIG. 15 shows H2B-GFP expression inthe extraembryonic tissue. FIG. 16 shows H2B-GFP expression inside ofthe zebra finch embryo, indicating that primordial germ cells carriedand expressed the transgene.

[0188] The eggs will hatch after approximately 20 days of incubationtime. Hatched chimeric chicks are raised to sexual maturity and thenmated. The eggs laid by the mated females are raised to hatching and theresulting transgenic chicks are identified, such as by Southern blot,PCR or expression analysis.

EXAMPLE 4

[0189] Transgenic fish may be made by the methods of the presentinvention. Breeding pairs of fish are placed in a water tank with agrooved bottom, where fertilized eggs are deposited. Fertilized eggs(zygotes) are collected and held in embryo medium on ice. Zygotes arealigned in grooves formed in a slab of agarose. A modified lentivirus,as described above, is loaded into a glass capillary micropipette. Thechorion membrane surrounding the zygote is pierced with the glassmicropipette and 200 nL of viral solution are delivered into the spacebetween the zygotic membrane and the chorion. Injected zygotes arereturned to a temperature-controlled water tank and allowed to mature.At sexual maturity, the founder fish are mated and their progenyanalyzed for the presence of the transgene, such as by Southern blot,PCR and protein analysis.

EXAMPLE 5

[0190] The modified lentivirus described above may also be used in genetrap experiments, such as in zebrafish. As discussed above, thistechnique allows the identification and cloning of a gene that isexpressed in a particular tissue or cell type and/or at a particulartime based solely on its pattern of expression. Zebrafish is an idealsystem for gene trapping for several reasons. First, embryonicdevelopment occurs externally, allowing for easy manipulation andviewing of the embryos. Furthermore, early stage zebrafish embryos aretranslucent, and the pigmentation can be further suppressed for severalmore days by incubating the embryo in a 0.003% solution of1-phenyl-2-thiourea (PTU). The translucent property of zebrafish embryosfacilitates the viewing of a live fluorescent reporter to identifytrapped genes expressed in spatial or temporal patterns of interest.

[0191] Self-inactivating lentiviral vectors are engineered to contain agene trap element consisting of the following sequences: spliceacceptor-IRES-GFP-poly A addition signal. This cassette is called SAIGP.The SAIGP element is inserted in a 3′ to 5′ orientation with respect tothe viral LTR sequences, to prevent inappropriate splicing ortermination of the viral genome during packaging. Zebrafish zygotes areinjected with VSVg-pseudotyped, concentrated SAIGP lentivirus asdescribed above. Fish are raised to sexual maturity and mated. Theprogeny are viewed with a fluorescent microscope, and GFP-expressingindividuals are separated for further analysis. GFP-positive animals arethen analyzed with a confocal fluorescent microscope to determine thespatial and temporal pattern of expression. Messenger RNA is extractedfrom those tissues of the animal that express GFP in the time and placeof interest, and reverse transcription with oligonucleotidescomplementary to GFP yields a cDNA that should contain the sequences ofthe trapped gene that flank the provirus. The recovered cDNA issubcloned into an appropriate bacterial plasmid, and the gene that hasbeen trapped by the SAIGFP provirus is identified by sequencing theupstream regions of the CDNA.

EXAMPLE 6

[0192] Virus particles generated from the FUGW vector were generated asdescribed above. The virus particles were injected using theperivitelline injection method, also described above, into 4 fertilizedrhesus monkey (Macaca mulatta) single cell embryos. Monkey oocytes canbe fertilized directly with sperm or can be fertilized using theintracytoplasmic sperm injection (ICSI) method. Of the four embryosinjected, 2 developed into blastocysts. Both blastocysts were green,evidencing expression of GFP. In the injected blastocysts, cells in thetrophectoder (TE) and the inner cell mass (ICM) both were green.Non-injected control embryos were not green. Transformed embryos aretransferred to host mothers for gestation. After approximately 150 to175 days, a newborn rhesus monkey is delivered which expresses GFPthroughout. Confirmation of the presence of the transgene and expressionin various tissues is carried out as described above.

1 9 1 178 DNA Human immunodeficiency virus 1 acaaatggca gtattcatccacaattttaa aagaaaaggg gggattgggg ggtacagtgc 60 aggggaaaga atagtagacataatagcaac agacatacaa actaaagaat tacaaaaaca 120 aattacaaaa attcaaaattttcgggttta ttacagggac agcagagatc cagtttgg 178 2 1221 DNA Homo sapiens 2gggtgcagcg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc ctcctcacgg 60cgagcgctgc cacgtcagac gaagggcgca ggagcgttcc tgatccttcc gcccggacgc 120tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat cagcagaagg 180acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca gagagcggaa 240caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg tggggcggtg 300aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg 360gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg agttgcgggc 420tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg 480agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg 540gggggagcgc acaaaatggc ggctgttccc gagtcttgaa tggaagacgc ttgtaaggcg 600ggctgtgagg tcgttgaaac aaggtggggg gcatggtggg cggcaagaac ccaaggtctt 660gaggccttcg ctaatgcggg aaagctctta ttcgggtgag atgggctggg gcaccatctg 720gggaccctga cgtgaagttt gtcactgact ggagaactcg ggtttgtcgt ctggttgcgg 780gggcggcagt tatgcggtgc cgttgggcag tgcacccgta cctttgggag cgcgcgcctc 840gtcgtgtcgt gacgtcaccc gttctgttgg cttataatgc agggtggggc cacctgccgg 900taggtgtgcg gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc 960tcctgaatcg acaggcgccg gacctctggt gaggggaggg ataagtgagg cgtcagtttc 1020tttggtcggt tttatgtacc tatcttctta agtagctgaa gctccggttt tgaactatgc 1080gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa atgtaatcat 1140ttgggtcaat atgtaatttt cagtgttaga ctagtaaatt gtccgctaaa ttctggccgt 1200ttttggcttt tttgttagac a 1221 3 720 DNA Artificial Sequence This sequenceencodes for a green fluorescent protein variant. 3 atggtgagca agggcgaggagctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtga acggccacaagttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagttcatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccttcacctacggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtccgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaactacaagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaagggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaacagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaagatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacacccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgccctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgccgccgggatc actcacggca tggacgagct gtacaagtaa 720 4 604 DNA Woodchuckhepatitis virus 4 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtattcttaactat 60 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatcatgctattgct 120 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtctctttatgag 180 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgctgacgcaacc 240 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggactttcgctttcccc 300 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctggacaggggct 360 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtcctttccatgg 420 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgctacgtcccttcg 480 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcggcctcttccg 540 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctccccgcctgat 600 cgat 604 5 1119 DNA Artificial Sequence This sequenceencodes for a fusion protein between human histone 2B protein and agreen fluorescent protein variant. 5 accatgccag agccagcgaa gtctgctcccgccccgaaaa agggctccaa gaaggcggtg 60 actaaggcgc agaagaaagg cggcaagaagcgcaagcgca gccgcaagga gagctattcc 120 atctatgtgt acaaggttct gaagcaggtccaccctgaca ccggcatttc gtccaaggcc 180 atgggcatca tgaattcgtt tgtgaacgacattttcgagc gcatcgcagg tgaggcttcc 240 cgcctggcgc attacaacaa gcgctcgaccatcacctcca gggagatcca gacggccgtg 300 cgcctgctgc tgcctgggga gttggccaagcacgccgtgt ccgagggtac taaggccatc 360 accaagtaca ccagcgctaa ggatccaccggtcgccacca tggtgagcaa gggcgaggag 420 ctgttcaccg gggtggtgcc catcctggtcgagctggacg gcgacgtaaa cggccacaag 480 ttcagcgtgt ccggcgaggg cgagggcgatgccacctacg gcaagctgac cctgaagttc 540 atctgcacca ccggcaagct gcccgtgccctggcccaccc tcgtgaccac cctgacctac 600 ggcgtgcagt gcttcagccg ctaccccgaccacatgaagc agcacgactt cttcaagtcc 660 gccatgcccg aaggctacgt ccaggagcgcaccatcttct tcaaggacga cggcaactac 720 aagacccgcg ccgaggtgaa gttcgagggcgacaccctgg tgaaccgcat cgagctgaag 780 ggcatcgact tcaaggagga cggcaacatcctggggcaca agctggagta caactacaac 840 agccacaacg tctatatcat ggccgacaagcagaagaacg gcatcaaggt gaacttcaag 900 atccgccaca acatcgagga cggcagcgtgcagctcgccg accactacca gcagaacacc 960 cccatcggcg acggccccgt gctgctgcccgacaaccact acctgagcac ccagtccgcc 1020 ctgagcaaag accccaacga gaagcgcgatcacatggtcc tgctggagtt cgtgaccgcc 1080 gccgggatca ctctcggcat ggacgagctgtacaagtaa 1119 6 1276 DNA Murine 6 tgagtcacca tgtgattgct gggaattgaactcaagacct ctggaagagc agtcagtgct 60 ctttttgttt gtttgtttgt ttgtttgtttgggttttttt tttgagacag ggtttctctg 120 tgtagccgtg tgtgtggggt agccctgtgtgtgagtgtgt gtgtgtgtgt ttgtggtatg 180 ttgcaaaata gattaaacaa ctgagagatggaataggtct tcttgacatc aaaaacatga 240 tcgtgaaccc ctttattaaa tctaacactcagagacagga gcatctctgc aggtttgagg 300 ccagcctgct ctacagaatg aatttcaggtctcaaggtca gcttggtcta caaagtgagt 360 ttcagatctc aaggccagcc aagactatgcagtaagacct tagctaaaat aaataaataa 420 ataaaataaa ataaaagtta atcttcgcttggcaaaccga taattgagga ccagtgctca 480 ggaaggaggc acacgggaat tccagaggctacagagggag cctcgctctg acctggttag 540 agcaactcta ctttactggc tgtgtctatgaggttctgct tgatttcatt tgacaaaaag 600 tttccacagc taaaccaggc aagggagccgaagtagacac agccacccgg gccgcgccca 660 acaggtttct ctctgctgct gagaagcaaaagcctgtttg aagaaactct ctgaaggaga 720 ctgtggttga gtggtggggg taggggtgctggggttgggc tgaggctgag ggttgactct 780 aaggagctgg aacctctcag cttcggtggctaggcagggg agttgtaatg aagagggaca 840 ggtaccctcc ttggtggagg agggtggaatgaaactctcg gtttccccca gaacttggca 900 aagtgtgtgt gatgtctccc aggtagtcccccaaaggagg aggctagcag agctggggag 960 gcaggaagtg ggtaactaga ctaacaaagatgcctgcctg tggcggtttg cccatcccag 1020 gtgggagggt gggactagcc ttgggcctgggcctcctgtg aacttggtgc ttgagggctc 1080 agagggaacc cagtcaggag cttgaatcccacgattcagc gcttctgtct gcggccaatg 1140 ggggcctctg agctgacgat ctcgggtactttttgtaact tccagaacag ggctctagga 1200 tgtctgatgt tggggcgagt ggcttagggccagctccttc aggcctctct acattccttc 1260 agggatcatg ggctgt 1276 7 1140 DNAMurine 7 gtctctagct gcatatgtag cagaagatgg cctagtcggc catcattgggaagagaggcc 60 ccttggtatt gcaaactata tgccccagta caggggaacg ccagggccaagaagtgggaa 120 tgagtgggta ggggagcagg gcggggggag ggggggttag ggaacttttgggatagcatt 180 tgaaatgtaa atgaagaaaa tatctaataa aaaataattt aaaaaagagcgtcagacagg 240 ggactgaaca gctcttgact aggggagaag aaggcaatgt agagtagtctgtgagttcta 300 atccttgcta aacactgact tcacctgacc cctactactt aaggcccccccccttactta 360 agaagtccct gtgttctctt acttcaatct acccccaaca tcatgagacctggtcaaaga 420 agctgtagaa acccaaaagt tgaatccatt tgcccttctg ggtttctgtctttgcctcca 480 tggacgatag ggacacacac acacacacac acacacacac acacacacacacgccccaaa 540 tctggagtgg tcctgatgtg gtagtggtag gtctttaggg gtctcatgggactgacatag 600 tatggtttaa ggtgctgctg agcaggaaag agaaggctaa gtggattttcaagacccctt 660 cccgtccgtc caagacaacc cctttcttgt tcccttcctg ccctgtccaccagctgcctt 720 ggaccatgga ggagagagta ggcaggaggc ccgggtagga gtaattgaaaggagcagatg 780 agacggggga atgcacccac ccccaccttc cctgccccac aggggctgtggagaaatgaa 840 aactaatcaa attacagccg acggcctccc gacccgtgca caggagccgcctgggccagg 900 ggcaggcctg cagggtgggg tgggggcaaa aggagaggga aggggaatcacatgtaacca 960 ctggaaacgt cttgatgtgc agcaacagct tagagggggg ctcaggtttctgtggcgttg 1020 gctatattta tctctgggtt catgccagca gggagggttt aaatggcacccagcagttgg 1080 tgtgaggggc tgcgggagct tgggggccag tggcaggaac aagccttttgcgacctgatg 1140 8 2853 DNA Artificial Sequence This sequence representsa vector insert comprising a human immunodeficiency virus sequence, agreen fluorescent protein variant sequence, a human ubiquitin promotersequence and a woodchuck hepatitis regulator element sequence. 8ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta 60cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca 120aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt 180ttggctgcag ttaattaaag atctgggtgc agcggcctcc gcgccgggtt ttggcgcctc 240ccgcgggcgc ccccctcctc acggcgagcg ctgccacgtc agacgaaggg cgcaggagcg 300ttcctgatcc ttccgcccgg acgctcagga cagcggcccg ctgctcataa gactcggcct 360tagaacccca gtatcagcag aaggacattt taggacggga cttgggtgac tctagggcac 420tggttttctt tccagagagc ggaacaggcg aggaaaagta gtcccttctc ggcgattctg 480cggagggatc tccgtggggc ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg 540cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg 600atcgtcactt ggtgagttgc gggctgctgg gctggccggg gctttcgtgg ccgccgggcc 660gctcggtggg acggaagcgt gtggagagac cgccaagggc tgtagtctgg gtccgcgagc 720aaggttgccc tgaactgggg gttgggggga gcgcacaaaa tggcggctgt tcccgagtct 780tgaatggaag acgcttgtaa ggcgggctgt gaggtcgttg aaacaaggtg gggggcatgg 840tgggcggcaa gaacccaagg tcttgaggcc ttcgctaatg cgggaaagct cttattcggg 900tgagatgggc tggggcacca tctggggacc ctgacgtgaa gtttgtcact gactggagaa 960ctcgggtttg tcgtctggtt gcgggggcgg cagttatgcg gtgccgttgg gcagtgcacc 1020cgtacctttg ggagcgcgcg cctcgtcgtg tcgtgacgtc acccgttctg ttggcttata 1080atgcagggtg gggccacctg ccggtaggtg tgcggtaggc ttttctccgt cgcaggacgc 1140agggttcggg cctagggtag gctctcctga atcgacaggc gccggacctc tggtgagggg 1200agggataagt gaggcgtcag tttctttggt cggttttatg tacctatctt cttaagtagc 1260tgaagctccg gttttgaact atgcgctcgg ggttggcgag tgtgttttgt gaagtttttt 1320aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt tagactagta 1380aattgtccgc taaattctgg ccgtttttgg cttttttgtt agacaaagct tctgcaggtc 1440gactctagag gatcccccgg gggtaccatg gtgagcaagg gcgaggagct gttcaccggg 1500gtggtgccca tcctggtcga gctggacggc gacgtgaacg gccacaagtt cagcgtgtcc 1560ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 1620ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg cgtgcagtgc 1680ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 1740ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 1800gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 1860aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 1920tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 1980atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 2040ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 2100cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 2160cacggcatgg acgagctgta caagtaagcg gccgctctag agaattcgat atcaagctta 2220tcgatatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta 2280actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta 2340ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt 2400atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg 2460caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt 2520tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag 2580gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc 2640catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc 2700cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc 2760ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc 2820ctgatcgata tcgatgtcga cctcgagggt acc 2853 9 9941 DNA Artificial SequenceThis sequence represents a viral vector having an insert comprising ahuman immunodeficiency virus sequence, a green fluorescent proteinvariant sequence, a human ubiquitin promoter sequence and a woodchuckhepatitis regulator element sequence. 9 gtcgacggat cgggagatct cccgatcccctatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctccctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggcaaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttcgcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaatcaattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacggtaaatggccc gcctggctga ccgcccaacg 360 acccccgccc attgacgtca ataatgacgtatgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttacggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattgacgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttatgggactttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggttttggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccaccccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtcgtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctatataagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggagctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgcttcaagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttttagtcagtgt ggaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaagggaaaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcgaggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggaggctag aaggagagagatgggtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattcggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcagggagctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaatactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatataatacagtagc aaccctctat tgtgtgcatc 1440 aaaggataga gataaaagac accaaggaagctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccgctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attatataaatataaagtag taaaaattga accattagga 1620 gtagcaccca ccaaggcaaa gagaagagtggtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagcagcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattgtctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctgttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaagatacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcaccactgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcacacgacctgga tggagtggga cagagaaatt 2040 aacaattaca caagcttaat acactccttaattgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaatgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattcataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtgaatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga cccacctccc aaccccgaggggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatccattcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtattcatccacaat tttaaaagaa aaggggggat 2460 tggggggtac agtgcagggg aaagaatagtagacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattcaaaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta agggtgcagcggcctccgcg ccgggttttg gcgcctcccg 2640 cgggcgcccc cctcctcacg gcgagcgctgccacgtcaga cgaagggcgc aggagcgttc 2700 ctgatccttc cgcccggacg ctcaggacagcggcccgctg ctcataagac tcggccttag 2760 aaccccagta tcagcagaag gacattttaggacgggactt gggtgactct agggcactgg 2820 ttttctttcc agagagcgga acaggcgaggaaaagtagtc ccttctcggc gattctgcgg 2880 agggatctcc gtggggcggt gaacgccgatgattatataa ggacgcgccg ggtgtggcac 2940 agctagttcc gtcgcagccg ggatttgggtcgcggttctt gtttgtggat cgctgtgatc 3000 gtcacttggt gagttgcggg ctgctgggctggccggggct ttcgtggccg ccgggccgct 3060 cggtgggacg gaagcgtgtg gagagaccgccaagggctgt agtctgggtc cgcgagcaag 3120 gttgccctga actgggggtt ggggggagcgcacaaaatgg cggctgttcc cgagtcttga 3180 atggaagacg cttgtaaggc gggctgtgaggtcgttgaaa caaggtgggg ggcatggtgg 3240 gcggcaagaa cccaaggtct tgaggccttcgctaatgcgg gaaagctctt attcgggtga 3300 gatgggctgg ggcaccatct ggggaccctgacgtgaagtt tgtcactgac tggagaactc 3360 gggtttgtcg tctggttgcg ggggcggcagttatgcggtg ccgttgggca gtgcacccgt 3420 acctttggga gcgcgcgcct cgtcgtgtcgtgacgtcacc cgttctgttg gcttataatg 3480 cagggtgggg ccacctgccg gtaggtgtgcggtaggcttt tctccgtcgc aggacgcagg 3540 gttcgggcct agggtaggct ctcctgaatcgacaggcgcc ggacctctgg tgaggggagg 3600 gataagtgag gcgtcagttt ctttggtcggttttatgtac ctatcttctt aagtagctga 3660 agctccggtt ttgaactatg cgctcggggttggcgagtgt gttttgtgaa gttttttagg 3720 caccttttga aatgtaatca tttgggtcaatatgtaattt tcagtgttag actagtaaag 3780 cttctgcagg tcgactctag aaaattgtccgctaaattct ggccgttttt ggcttttttg 3840 ttagacagga tccccgggta ccggtcgccaccatggtgag caagggcgag gagctgttca 3900 ccggggtggt gcccatcctg gtcgagctggacggcgacgt aaacggccac aagttcagcg 3960 tgtccggcga gggcgagggc gatgccacctacggcaagct gaccctgaag ttcatctgca 4020 ccaccggcaa gctgcccgtg ccctggcccaccctcgtgac caccctgacc tacggcgtgc 4080 agtgcttcag ccgctacccc gaccacatgaagcagcacga cttcttcaag tccgccatgc 4140 ccgaaggcta cgtccaggag cgcaccatcttcttcaagga cgacggcaac tacaagaccc 4200 gcgccgaggt gaagttcgag ggcgacaccctggtgaaccg catcgagctg aagggcatcg 4260 acttcaagga ggacggcaac atcctggggcacaagctgga gtacaactac aacagccaca 4320 acgtctatat catggccgac aagcagaagaacggcatcaa ggtgaacttc aagatccgcc 4380 acaacatcga ggacggcagc gtgcagctcgccgaccacta ccagcagaac acccccatcg 4440 gcgacggccc cgtgctgctg cccgacaaccactacctgag cacccagtcc gccctgagca 4500 aagaccccaa cgagaagcgc gatcacatggtcctgctgga gttcgtgacc gccgccggga 4560 tcactctcgg catggacgag ctgtacaagtaaagcggccg cgactctaga attcgatatc 4620 aagcttatcg ataatcaacc tctggattacaaaatttgtg aaagattgac tggtattctt 4680 aactatgttg ctccttttac gctatgtggatacgctgctt taatgccttt gtatcatgct 4740 attgcttccc gtatggcttt cattttctcctccttgtata aatcctggtt gctgtctctt 4800 tatgaggagt tgtggcccgt tgtcaggcaacgtggcgtgg tgtgcactgt gtttgctgac 4860 gcaaccccca ctggttgggg cattgccaccacctgtcagc tcctttccgg gactttcgct 4920 ttccccctcc ctattgccac ggcggaactcatcgccgcct gccttgcccg ctgctggaca 4980 ggggctcggc tgttgggcac tgacaattccgtggtgttgt cggggaaatc atcgtccttt 5040 ccttggctgc tcgcctgtgt tgccacctggattctgcgcg ggacgtcctt ctgctacgtc 5100 ccttcggccc tcaatccagc ggaccttccttcccgcggcc tgctgccggc tctgcggcct 5160 cttccgcgtc ttcgccttcg ccctcagacgagtcggatct ccctttgggc cgcctccccg 5220 catcgatacc gtcgacctcg agacctagaaaaacatggag caatcacaag tagcaataca 5280 gcagctacca atgctgattg tgcctggctagaagcacaag aggaggagga ggtgggtttt 5340 ccagtcacac ctcaggtacc tttaagaccaatgacttaca aggcagctgt agatcttagc 5400 cactttttaa aagaaaaggg gggactggaagggctaattc actcccaacg aagacaagat 5460 atccttgatc tgtggatcta ccacacacaaggctacttcc ctgattggca gaactacaca 5520 ccagggccag ggatcagata tccactgacctttggatggt gctacaagct agtaccagtt 5580 gagcaagaga aggtagaaga agccaatgaaggagagaaca cccgcttgtt acaccctgtg 5640 agcctgcatg ggatggatga cccggagagagaagtattag agtggaggtt tgacagccgc 5700 ctagcatttc atcacatggc ccgagagctgcatccggact gtactgggtc tctctggtta 5760 gaccagatct gagcctggga gctctctggctaactaggga acccactgct taagcctcaa 5820 taaagcttgc cttgagtgct tcaagtagtgtgtgcccgtc tgttgtgtga ctctggtaac 5880 tagagatccc tcagaccctt ttagtcagtgtggaaaatct ctagcagggc ccgtttaaac 5940 ccgctgatca gcctcgactg tgccttctagttgccagcca tctgttgttt gcccctcccc 6000 cgtgccttcc ttgaccctgg aaggtgccactcccactgtc ctttcctaat aaaatgagga 6060 aattgcatcg cattgtctga gtaggtgtcattctattctg gggggtgggg tggggcagga 6120 cagcaagggg gaggattggg aagacaatagcaggcatgct ggggatgcgg tgggctctat 6180 ggcttctgag gcggaaagaa ccagctggggctctaggggg tatccccacg cgccctgtag 6240 cggcgcatta agcgcggcgg gtgtggtggttacgcgcagc gtgaccgcta cacttgccag 6300 cgccctagcg cccgctcctt tcgctttcttcccttccttt ctcgccacgt tcgccggctt 6360 tccccgtcaa gctctaaatc gggggctccctttagggttc cgatttagtg ctttacggca 6420 cctcgacccc aaaaaacttg attagggtgatggttcacgt agtgggccat cgccctgata 6480 gacggttttt cgccctttga cgttggagtccacgttcttt aatagtggac tcttgttcca 6540 aactggaaca acactcaacc ctatctcggtctattctttt gatttataag ggattttgcc 6600 gatttcggcc tattggttaa aaaatgagctgatttaacaa aaatttaacg cgaattaatt 6660 ctgtggaatg tgtgtcagtt agggtgtggaaagtccccag gctccccagc aggcagaagt 6720 atgcaaagca tgcatctcaa ttagtcagcaaccaggtgtg gaaagtcccc aggctcccca 6780 gcaggcagaa gtatgcaaag catgcatctcaattagtcag caaccatagt cccgccccta 6840 actccgccca tcccgcccct aactccgcccagttccgccc attctccgcc ccatggctga 6900 ctaatttttt ttatttatgc agaggccgaggccgcctctg cctctgagct attccagaag 6960 tagtgaggag gcttttttgg aggcctaggcttttgcaaaa agctcccggg agcttgtata 7020 tccattttcg gatctgatca gcacgtgttgacaattaatc atcggcatag tatatcggca 7080 tagtataata cgacaaggtg aggaactaaaccatggccaa gttgaccagt gccgttccgg 7140 tgctcaccgc gcgcgacgtc gccggagcggtcgagttctg gaccgaccgg ctcgggttct 7200 cccgggactt cgtggaggac gacttcgccggtgtggtccg ggacgacgtg accctgttca 7260 tcagcgcggt ccaggaccag gtggtgccggacaacaccct ggcctgggtg tgggtgcgcg 7320 gcctggacga gctgtacgcc gagtggtcggaggtcgtgtc cacgaacttc cgggacgcct 7380 ccgggccggc catgaccgag atcggcgagcagccgtgggg gcgggagttc gccctgcgcg 7440 acccggccgg caactgcgtg cacttcgtggccgaggagca ggactgacac gtgctacgag 7500 atttcgattc caccgccgcc ttctatgaaaggttgggctt cggaatcgtt ttccgggacg 7560 ccggctggat gatcctccag cgcggggatctcatgctgga gttcttcgcc caccccaact 7620 tgtttattgc agcttataat ggttacaaataaagcaatag catcacaaat ttcacaaata 7680 aagcattttt ttcactgcat tctagttgtggtttgtccaa actcatcaat gtatcttatc 7740 atgtctgtat accgtcgacc tctagctagagcttggcgta atcatggtca tagctgtttc 7800 ctgtgtgaaa ttgttatccg ctcacaattccacacaacat acgagccgga agcataaagt 7860 gtaaagcctg gggtgcctaa tgagtgagctaactcacatt aattgcgttg cgctcactgc 7920 ccgctttcca gtcgggaaac ctgtcgtgccagctgcatta atgaatcggc caacgcgcgg 7980 ggagaggcgg tttgcgtatt gggcgctcttccgcttcctc gctcactgac tcgctgcgct 8040 cggtcgttcg gctgcggcga gcggtatcagctcactcaaa ggcggtaata cggttatcca 8100 cagaatcagg ggataacgca ggaaagaacatgtgagcaaa aggccagcaa aaggccagga 8160 accgtaaaaa ggccgcgttg ctggcgtttttccataggct ccgcccccct gacgagcatc 8220 acaaaaatcg acgctcaagt cagaggtggcgaaacccgac aggactataa agataccagg 8280 cgtttccccc tggaagctcc ctcgtgcgctctcctgttcc gaccctgccg cttaccggat 8340 acctgtccgc ctttctccct tcgggaagcgtggcgctttc tcatagctca cgctgtaggt 8400 atctcagttc ggtgtaggtc gttcgctccaagctgggctg tgtgcacgaa ccccccgttc 8460 agcccgaccg ctgcgcctta tccggtaactatcgtcttga gtccaacccg gtaagacacg 8520 acttatcgcc actggcagca gccactggtaacaggattag cagagcgagg tatgtaggcg 8580 gtgctacaga gttcttgaag tggtggcctaactacggcta cactagaaga acagtatttg 8640 gtatctgcgc tctgctgaag ccagttaccttcggaaaaag agttggtagc tcttgatccg 8700 gcaaacaaac caccgctggt agcggtggtttttttgtttg caagcagcag attacgcgca 8760 gaaaaaaagg atctcaagaa gatcctttgatcttttctac ggggtctgac gctcagtgga 8820 acgaaaactc acgttaaggg attttggtcatgagattatc aaaaaggatc ttcacctaga 8880 tccttttaaa ttaaaaatga agttttaaatcaatctaaag tatatatgag taaacttggt 8940 ctgacagtta ccaatgctta atcagtgaggcacctatctc agcgatctgt ctatttcgtt 9000 catccatagt tgcctgactc cccgtcgtgtagataactac gatacgggag ggcttaccat 9060 ctggccccag tgctgcaatg ataccgcgagacccacgctc accggctcca gatttatcag 9120 caataaacca gccagccgga agggccgagcgcagaagtgg tcctgcaact ttatccgcct 9180 ccatccagtc tattaattgt tgccgggaagctagagtaag tagttcgcca gttaatagtt 9240 tgcgcaacgt tgttgccatt gctacaggcatcgtggtgtc acgctcgtcg tttggtatgg 9300 cttcattcag ctccggttcc caacgatcaaggcgagttac atgatccccc atgttgtgca 9360 aaaaagcggt tagctccttc ggtcctccgatcgttgtcag aagtaagttg gccgcagtgt 9420 tatcactcat ggttatggca gcactgcataattctcttac tgtcatgcca tccgtaagat 9480 gcttttctgt gactggtgag tactcaaccaagtcattctg agaatagtgt atgcggcgac 9540 cgagttgctc ttgcccggcg tcaatacgggataataccgc gccacatagc agaactttaa 9600 aagtgctcat cattggaaaa cgttcttcggggcgaaaact ctcaaggatc ttaccgctgt 9660 tgagatccag ttcgatgtaa cccactcgtgcacccaactg atcttcagca tcttttactt 9720 tcaccagcgt ttctgggtga gcaaaaacaggaaggcaaaa tgccgcaaaa aagggaataa 9780 gggcgacacg gaaatgttga atactcatactcttcctttt tcaatattat tgaagcattt 9840 atcagggtta ttgtctcatg agcggatacatatttgaatg tatttagaaa aataaacaaa 9900 taggggttcc gcgcacattt ccccgaaaagtgccacctga c 9941

What is claimed is:
 1. A method of producing a transgenic birdcomprising: transfecting a packaging cell line with a retroviralconstruct; recovering recombinant retroviral particles from thepackaging cell line; and infecting a bird egg with the recombinantretroviral particles, wherein the retroviral construct comprises the Rand U5 sequences from a 5′ lentiviral LTR and a self-inactivating 3′lentiviral LTR.
 2. The method of claim 1 wherein infecting a bird eggcomprises contacting the embryonic blastodisc of the bird egg with theretroviral particles.
 3. The method of claim 1 wherein said retroviralconstruct additionally comprises an internal promoter.
 4. The method ofclaim 1 wherein said retroviral construct additionally comprises a geneof interest.
 5. The method of claim 4 wherein said transgenic animalexpresses the gene of interest.
 6. The method of claim 1 wherein saidpackaging cell line is a 293 cell line.
 7. The method of claim 1 whereinthe 5′ LTR sequences are from HIV.
 8. The method of claim 1 wherein theself-inactivating 3′ LTR comprises a U3 element with a deletion of itsenhancer sequence.
 9. The method of claim 8 wherein theself-inactivating 3′ LTR is a modified HIV 3′ LTR.
 10. The method ofclaim 1 wherein the recombinant retrovirus is pseudotyped.
 11. Themethod of claim 10 wherein the recombinant retrovirus is pseudotypedwith the vesicular stomatitits virus envelope glycoprotein.
 12. Themethod of claim 1 wherein the viral construct additionally comprises apromoter operably linked to the R and U5 5′ LTR sequences.
 13. Themethod of claim 12 wherein the promoter is a CMV promoter.
 14. Themethod of claim 12 wherein the viral construct additionally comprises anenhancer operably linked to the promoter.
 15. The method of claim 14wherein the enhancer and promoter are CMV sequences.
 16. The method ofclaim 1 wherein the viral construct additionally comprises the woodchuckhepatitis virus enhancer element sequence.
 17. The method of claim 1wherein the viral construct additionally comprises a tRNA ambersuppressor sequence.
 18. The method of claim 3 wherein the viralconstruct additionally comprises a reporter gene operably linked to theinternal promoter.
 19. The method of claim 18 wherein the reporter geneencodes a fluorescent protein.
 20. The method of claim 19 wherein saidfluorescent protein is green fluorescent protein.
 21. The method ofclaim 3 wherein the internal promoter is a ubiquitous promoter.
 22. Themethod of claim 21 wherein said ubiquitous promoter is selected from thegroup consisting of the ubiquitin promoter, the CMV β-actin promoter andthe pgk promoter.
 23. The method of claim 3 wherein the internalpromoter is a tissue specific promoter.
 24. The method of claim 23wherein said tissue specific promoter is selected from the groupconsisting of the lck promoter, the myogenin promoter and the thy1promoter.
 25. A transgenic bird made by the method of claim
 1. 26. Amethod of producing a transgenic bird comprising the following steps: a)opening a window in the shell of a fertilized bird egg; b) injectingmodified retrovirus into the space between the perivitelline membraneand the embryonic blastodisc; and c) incubating the embryo untilhatching.
 27. The method of claim 26 wherein the modified retrovirus isa modified lentivirus.
 28. The method of claim 27 wherein the modifiedlentivirus is produced by transfecting a packaging cell line with aviral construct.
 29. The method of claim 28 wherein the viral constructcomprises the R and U5 sequences from a lentiviral 5′ LTR, an internalpromoter, a gene of interest, and a self-inactivating lentiviral 3′ LTR.30. A transgenic bird wherein one or more germ cells comprises proviralDNA, said proviral DNA comprising a self-inactivating 3′ lentiviral LTR.31. The transgenic bird of claim 30 wherein the self-inactivating 3′ LTRis a self-inactivating 3′ HIV LTR.
 32. The transgenic bird of claim 30wherein the self-inactivating 3′ HIV LTR comprises a U3 element with adeletion of its enhancer sequence.
 33. A method of producing atransgenic fish comprising: transfecting a packaging cell line with aviral construct; recovering recombinant retroviral particles from thepackaging cell line; and infecting a fish egg with the recombinantretroviral particles, wherein the viral construct comprises the R and U5sequences from a 5′ lentiviral LTR and a self-inactivating 3′ lentiviralLTR.
 34. The method of claim 33 wherein infecting a fish egg comprisesdelivering the retroviral particles to the space between the chorion andthe cell membrane of the fish egg.
 35. The method of claim 33 whereinsaid retroviral construct additionally comprises an internal promoter.36. The method of claim 33 wherein said retroviral constructadditionally comprises a gene of interest.
 37. The method of claim 37wherein said transgenic animal expresses the gene of interest.
 38. Atransgenic fish made by the method of claim
 33. 39. A transgenic fishwhose genome comprises proviral DNA, said proviral DNA comprising aself-inactivating 3′ lentiviral LTR.
 40. The transgenic fish of claim 39wherein the self-inactivating 3′ LTR is a self-inactivating 3′ HIV LTR.41. The transgenic fish of claim 39 wherein the self-inactivating 3′ HIVLTR comprises a U3 element with a deletion of its enhancer sequence.